IP-to-MS:
An Unbiased Workflow for Antigen Profiling
Immunoprecipitation
(IP) is one of the
most widely used methods
in biological and medical research. The
breadth of IP applications is staggering, ranging from demonstration
of cell/tissue-specific antigens to identifying associated coimmunoprecipitates,
and from detection of viral antigens in patient samples to characterization
of disease-specific autoantibodies in organ-based versus systemic
autoimmune disease. Traditionally, the primary end point of a conventional
IP assay has been gel electrophoresis to visualize the presence of
the antigen and associated proteins. Mass spectrometry (MS)
has become vital for identifying target
antigens and associated proteins in the above-mentioned applications. − However, the presence of overwhelming levels of immunoglobulins
presents a challenge for MS of immunoprecipitates. To facilitate MS
of immunoprecipitated proteins, antibodies are either bound to protein
A/protein G resin, or covalently cross-linked to bead-based matrices. , The bound target and associated proteins, plus nonspecifically bound
proteins and leached immunoglobulins, are eluted from the resin and
separated by gel electrophoresis. Bands of interest are manually excised
from the gel and processed by in-gel trypsin digestion. The resultant
peptides are typically cleaned up by tip-based solid support columns
and analyzed by liquid chromatography (LC)–MS. This is a complex,
multistep process that requires several days of processing, significant
amounts of input antibody, and advanced technical expertise. Current IP–MS methods are plagued by immunoglobulin contamination
and high background noise due to nonspecifically bound proteins. While
cross-linking of antibodies to bead-based matrices reduces immunoglobulin
contamination, these treatments require optimization to balance adequate
antibody coupling with potential antibody inactivation caused by excess
cross-linking reagent. The final outputs of these IP–MS experiments
are relatively long lists of candidate proteins that require further
study to differentiate between true target candidates and nonspecifically
bound proteins. , Patient-specific autoantibody/autoantigen
discovery is among the
most challenging applications of IP. Experiments designed for this
purpose require patient serum or plasma (which is often in limited
supply) and different amounts of tissue or cells to generate substrate
antigens. The typical path to identifying new autoantigens begins
with binding of patient antibodies to protein A resin and IP of radiolabeled
protein from cell lysates. − Radiolabeling of target proteins allows one to discriminate
immunoprecipitated target proteins from immunoglobulin protein when
separated by gel electrophoresis and visualized by autoradiography.
Because radioactive proteins cannot be safely analyzed by MS, the
autoradiograph is overlaid on a gel containing preparative amounts
of immunoprecipitate. The matching bands are excised from the preparative
IP gel, in-gel digested with trypsin, and subsequently analyzed by
LC–MS. Serological proteome analysis is a similar approach
which has been used to identify antigens in various diseases. − While these methods are proven, they are labor intensive and technically
demanding processes that cannot practically be applied to large numbers
of patients. As a result, current approaches for patient-specific
autoantibody/autoantigen
identification rely on a relatively small set of common autoantigens
that can be used as standards for assessing targets of patient antibodies.
Methods to detect relevant autoantibodies include enzyme-linked immunosorbent
assay (ELISA), Ouchterlony double immunodiffusion (DID), and IP of
radioactively tagged target proteins. These are all targeted assays
that rely on previously identified autoantigens characterized by large-scale
studies of common autoimmune diseases. Thus, they do not afford the
opportunity to identify novel autoantibody specificities in rare autoimmune
diseases lacking defined molecular characterization. Protein
microarrays provide another method to identify autoantibodies. While these allow for multiplex searches and
offer a wider variety of potential autoantigen targets, they have
certain shortcomings. First, they are biased in the number of potential
autoantigen targets. Second, since these proteins are typically produced
by microorganisms or in vitro, they do not carry the same post-translational
modifications seen in human cells, their folded state is not certain,
and their relative abundances do not match that of human cells, all
of which impact antibody binding affinity. Thus, a truly unbiased
population of putative autoantigens should come from human cells or
tissues, with their typical folded structures, modifications, and
abundances. In the context of autoimmune
diseases that impact more than 20
million individuals in the US, roughly 30% do not have an identifiable
autoantigen target using conventional methods of antibody detection. − Therefore, there is a critical need for improved “agnostic”
technological approaches that promote autoantigen discovery. To advance
the methodology for identification and characterization of antigen/autoantigen
targets, we have devised a novel, radiolabel-free method, referred
to as IP-to-MS, that allows direct MS identification of antigens derived
from any cell or tissue source that are recognized by antibodies found
in the sera of individual patients. The method we describe here provides
a means to isolate antigen targets that are largely free of immunoglobulins,
allowing in situ digestion into tryptic peptides and subsequent MS
analysis. In this method, we have employed a protein capture
reagent, ProMTag,
to replace radiolabeling in separating immunoprecipitated antigens
from immunoglobulins in IP eluates. The ProMTag is composed of three
elements: a reversible protein-binding moiety that couples to amine
groups on proteins in a pH-dependent manner; a flexible polyethylene
glycol linker; and a methyltetrazine (MT) group for rapid, irreversible
coupling to trans -cyclooctene (TCO), which is a well-known
click chemistry pair. , Although the ProMTag was
originally developed for whole proteome
capture and release, we have adapted
this technology for unbiased IP of target autoantigens. This approach
involving ProMTag demonstrates several critical features—namely,
that binding of ProMTagged protein to TCO resin facilitates removal
of the vast majority of serum immunoglobulins, and that ProMTagged
proteins are amenable to release as intact proteins suitable for gel
electrophoresis and/or MS identification. We demonstrate that the
patient-specific IP-to-MS workflow allows for the rapid identification
of novel autoantigens targeted by both previously characterized patient
sera and sera previously designated as “seronegative”
based on available clinical assays. Furthermore, we show that IP-to-MS
can rival or surpass traditional methods of antibody detection, such
as ELISA and Ouchterlony DID. Because we are presenting a novel,
unbiased approach to identifying
putative autoantigens, we have devised slightly modified nomenclature
to describe the output of this methodological approach. Rather than
disease-specific antibodies, we refer to the output as patient-specific
antigens. This designation indicates that the analysis is directed
to a single individual, as opposed to a particular disease or patient
population. Importantly, we do not use patient-matched lysates as
the antigen sources for the experiments here; therefore, in these
IP-to-MS assays the autoantibodies, but not the autoantigens, are
patient-specific. Because the IP-to-MS method produces lists of putative
target proteins, rather than antibodies per-se, we will refer to the
output of the IP-to-MS method as potential autoantigens. The goal
of employing IP-to-MS in this test case of analyzing sera from patients
with autoimmune disease is to enable large-scale, quantitative data
collection and analysis that will advance autoimmune disease classification. 

Cell Lysate and Sera Sources K562 cells were grown
by Cell Culture Company. We used the erythroleukemoid cell line K562
because these cells grow rapidly in culture and, although not tissue-specific,
express a wide range of proteins that include commonly targeted autoantigens.
As such, these cells represent the gold standard in the field of IP/autoantibody
detection. To prepare K562 cell lysates, cells were suspended
in IP lysis buffer (IP-LB; Impact Proteomics, LLC.). The cells were
sonicated on ice for 30–40 pulses at 30% power and 30% duty
cycle on a Branson 450 Sonifier. The sample was centrifuged at 14,000
rpm for 20 min in a refrigerated benchtop centrifuge at 4 °C.
The supernatant was removed, and the lysate was stored in 500 μL
aliquots at −80 °C. Protein concentration was determined
via a Pierce BCA assay (Thermo Fisher Scientific). Patient-derived
samples utilized in experiments shown in – were collected
as part of two separate longitudinal
registries at the University of Pittsburgh encompassed by IRB-approved
protocols 20030223 and 19090054. All patients (excluding healthy controls)
had interstitial lung disease associated with an underlying connective
tissue disease such as scleroderma, idiopathic inflammatory myopathy,
or systemic lupus erythematosus. ProMTag Titration to Assess
Protein Capture and Release Six samples of 50 μg of
K562 lysate were brought to a concentration
of 0.5 mg/mL with IP-LB. Varying amounts of 30 mg/mL ProMTag was added
(0, 0.25, 0.5, 1.0, 3.0, or 5.0 μL); 100% acetonitrile (ACN)
(5.0, 4.75, 4.5, 4.0, 2.0, or 0 μL) was added to bring each
sample to a final volume of 55 μL. Samples were incubated on
ice for 30 min, then 10 μL of Quencher (Impact Proteomics, LLC.)
was added to each sample to quench the labeling reaction. The samples
were incubated on ice for another 30 min. Samples were then incubated
at 4 °C with rotation for 2 h. After the 2 h incubation
was complete, 20% sodium dodecyl sulfate (SDS) (Bio-Rad) was added
to a final concentration of 1.07% and the samples were incubated at
room temperature with rotation for 15 min. During this incubation,
100 μL TCO agarose resin (Vector Laboratories, Inc.) per sample
was washed once with water in resin capture (RC) tubes, which have
a small slit in the bottom that allows the passing of liquid with
minimal dead volume, but retains the solid resin (Impact Proteomics,
LLC.). The samples were added to the TCO resin and incubated at room
temperature with rotation for 15 min. The flowthrough was collected
by brief centrifugation of the RC
tubes. The resin was then washed twice with 200 μL 100 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid) (HEPES), 1% SDS and once with 200 μL 1 mM HEPES, 1% SDS.
Each wash was collected in separate 1.5 mL tubes. To elute the
protein from the TCO resin, 75 μL of 100 mM
formic acid (FA), 1% SDS was added followed by a 10 min incubation
at room temperature with rotation. The elution step was repeated once,
with both eluates being collected into the same 1.5 mL tube by brief
centrifugation of the RC tubes. For each sample, the flowthrough
was combined with the first wash.
The flowthrough + wash 1 fraction and the eluate were dried fully
in a SpeedVac. The flowthrough + wash 1 fraction was resuspended in
100 μL water, and the eluate was resuspended in 100 μL
100 mM HEPES pH 8.0. The samples were run on 4–20% SDS-polyacrylamide
gel electrophoresis (PAGE) gels (Bio-Rad) for 1.5 h at 120 V and the
gels were stained with Coomassie Brilliant Blue R-250 (Bio-Rad). Quantification
of the SDS-PAGE gels was done with ImageJ, with the blank region between
the third and fourth lanes used for background subtraction. The percent
of protein remaining in the flowthrough + wash 1 or the percent of
protein in the eluate was quantified according to the pixel intensity
of the gel images using ImageJ. The protein amount was calculated
as a percentage of the “0 ProMTag” control for the “flowthrough
+ wash 1” gel or as a percentage of the 12.5 μg ProMTag
per μg lysate sample for the “eluate” gel. Removal
of Immunoglobulin before and after TCO Resin Elution HeLa
lysate in IP-LB was labeled with ProMTag for 30 min at room
temperature at a ratio of 1.67 μg of protein per microgram of
ProMTag. The labeling reaction was quenched by addition of Quencher
followed by a 30 min incubation at room temperature. During
labeling of HeLa lysate, protein A resin was prepared as follows:
5 μL HiTrap MabSelect PrismA resin (Cytiva Life Sciences) was
added to a RC tube and washed 3 times with 300 μL IPP buffer
(Impact Proteomics, LLC.). The RC tube was sealed and the PrismA resin
was resuspended in 25 μL IPP, then 2.5 μL polyclonal anti-Hsp90
(Abcam) and 10 μL patient serum with anti-Topo I autoantibody
was added. Antibody binding to the PrismA resin was carried out for
1.5 h at room temperature with end-over-end rotation. The resin was
then washed 4 times with 300 μL phosphate-buffered saline (PBS)
and 3 times with 300 μL IPP. The resin was suspended in 50 μL
IPP, then CyDye Cy5-NHS minimal dye (Cytiva) was added to a concentration
of 20 μM and labeling was carried out for 15 min at room temperature.
The labeling reaction was quenched by addition of Quencher followed
by a 15 min incubation at room temperature. The resin was washed 2
times with 300 μL IPP and the RC tube was sealed with a stopper
that prevents liquid flow. The ProMTagged HeLa lysate (250 μL)
was then added to the
prepared PrismA resin. The resin was incubated with the lysate for
2 h at room temperature. The resin was then washed 4 times with 300
μL IPP. To elute proteins from the PrismA resin, 50 μL
100 mM HEPES pH 8.0, 1% SDS was added to the resin followed by a 10
min incubation at room temperature. This elution step was repeated
once with both eluates for each sample being collected in the same
1.5 mL tube. One-third of the PrismA resin eluate (33 μL)
was used as
the Load sample. This fraction, which contained Cy5-labeled proteins
that had been bound and eluted from the PrismA beads (mostly Cy5-immunoglobulins),
was labeled with CyDye Cy3-NHS at 20 μM concentration for 15
min at room temperature and then quenched by adding quencher followed
by a 15 min incubation at room temperature. The second third of the
PrismA eluate was added to 20 μL TCO resin that had been washed
twice with 300 μL IP-WB3 in a RC tube. The TCO resin was incubated
at room temperature for 30 min. The protein fraction that did not
bind to the TCO beads was saved as the flowthrough fraction. The flowthrough
fraction, which also contained Cy5-labeled proteins that had been
bound and eluted from the PrismA beads (mostly Cy5-immunoglobulins),
was labeled with CyDye Cy3-NHS at 20 μM concentration for 15
min at room temperature and quenched by the addition of quencher,
as described above. The final third of the PrismA eluate was
added to 20 μL TCO
resin that had been washed twice with 300 μL IP-WB3 in a RC
tube. The TCO resin was washed 3 times with 300 μL 100 mM HEPES
pH 8.0, 1% SDS, 10 mM dl -dithiothreitol (DTT); after the
first addition of this buffer only, the sample was incubated with
mixing for 30 min at room temperature. The TCO resin was then washed
an additional 2 times with 300 μL 100 mM HEPES pH 8.0, then
resuspended in 50 μL 100 mM HEPES pH 8.0. Protein bound to the
TCO resin was labeled with CyDye Cy3-NHS minimal dye (20 μM)
for 15 min at room temperature before being quenched by addition of
quencher followed by a 15 min incubation at room temperature. The
TCO resin was washed once with 300 μL 100 mM HEPES pH 8.0, then
once with 300 μL 1% SDS. To elute protein from the TCO resin,
33 μL 100 mM FA, 1% SDS was added followed by a 30 min incubation
at 37 °C. The Eluate fraction was collected and neutralized by
addition of 1 M tetraethylammonium bromide (TEAB). Samples were
prepared for SDS-PAGE as follows: 33 μL of the
load, flowthrough, and eluate fractions were mixed with 11 μL
4× sample buffer (Bio-Rad). Samples were run on a 4–20%
SDS-PAGE gel (Bio-Rad) for ∼1.5 h at 120 V until the dye front
had completely run out of the gel. Fluorescence images were acquired
using a custom-built imager. IP-to-MS of
37 Patient Sera Sera antibodies were bound
to rProtein A Sepharose Fast Flow resin (Cytiva) ( , step 1B). Twenty microliters
rProtein A resin was washed twice with 200 μL IPP buffer in
RC tubes. The RC tubes were sealed, then 30 μL IPP buffer was
added to the rProtein A resin. Next, 20 μL serum was added to
each tube, followed by a 45 min incubation at 4 °C with rotation.
After this incubation, the resin was washed four times with 300 μL
IPP buffer and the RC tubes were sealed. During binding of antibodies to rProtein A resin,
K562 lysate was
brought to a concentration of 0.5 mg/mL in IP-LB. ProMTag was added
to the lysate at a ratio of 1.8 μg of ProMTag per microgram
of protein, and the labeling reaction was incubated for 30 min on
ice ( , step
1A). The labeling reaction was quenched by addition of 20 μL
quencher per 200 μg lysate followed by a 30 min incubation on
ice. The ProMTagged K562 lysate (200 μg per sample) was
then added
to the prepared rProtein A resin ( , “Bind ProMTagged target proteins
to Ig on protein A resin”). The samples were incubated at 4
°C for 2 h with rotation. The resin was then washed four times
with 300 μL IPP buffer ( , “wash away unbound ProMTagged proteins”).
To elute proteins from the rProtein A resin, 25 μL IP-elution
buffer (IP-EB; Impact Proteomics, LLC.) was added to the resin followed
by a 10 min incubation at room temperature with rotation ( , “elute ProMTagged
target proteins & Ig”). This elution step was repeated
once with both eluates for each sample being collected in the same
1.5 mL tube. The eluates from the rProtein A resin were then
added to 20 μL
TCO resin that had been washed with 300 μL IP-wash buffer 3
(IP-WB3; Impact Proteomics, LLC.) in an RC tube ( , “add TCO resin”).
The samples were incubated for 15 min at room temperature with rotation.
The TCO resin was then washed as follows: three times with 300 μL
IP-EB, three times with 300 μL IP-Wash Buffer 1 (IP-WB1; Impact
Proteomics, LLC.), two times with 300 μL IP-Wash Buffer 2 (IP-WB2;
Impact Proteomics, LLC.), one time with 300 μL IP-WB3, and two
times with 300 μL ultrapure water ( , “wash away unbound Ig”). Next, 40 μL 100 mM FA was added to the TCO resin ( , “release
target proteins”) followed by 20 μL MT-Trypsin (Impact
Proteomics, LLC.). Samples were incubated at 37 °C for 1 h. The
eluate was collected by brief centrifugation of the RC tube, then
an additional 40 μL 100 mM FA was added to the TCO resin. Samples
were incubated at room temperature with rotation for 15 min. The RC
tubes were once again briefly centrifuged into the same 1.5 mL tube
as for the previous eluate ( , “elute target protein peptides ready for MS”).
The eluates were then dried fully in a SpeedVac and stored at −80
°C. MS Analysis of Patient Autoantigens Each sample was
desalted using an Evotip Pure C18 disposable tip (EV2011, Evosep)
following the manufacturer’s protocol with the following modifications:
(1) for conditioning, instead of soaking the tips in 1-propanol, the
Evotips were washed with 20 μL of 2-propanol (Optima LC/MS grade,
Fisher Chemical) and centrifuged at 700 g for 45–50
s until the solvent level was within 1 mm above the packing material;
(2) the centrifugation steps for rinsing, equilibration, and washing
were shortened to 10–15 s; (3) both the rinse and wash steps
were repeated twice with 20 μL 0.1% FA in water (Optima LC/MS
grade, Fisher Chemical). With an Evosep One HPLC (Evosep), the
desalted peptides were eluted off of the Evotip and loaded on to an
Evosep EV1109 performance analytical column (8 cm × 150 μm
inner diameter, 1.5 μm ReproSil Saphir C18 beads from Dr. Maisch).
Peptide separation was carried out according to the manufacturer’s
preset 11.5 min, 100 samples-per-day (SPD), method with 0.1% FA in
water as solvent A and 0.1% FA in ACN as solvent B (Optima LC/MS grade,
Fisher Chemical). All mass spectrometric data were collected
with a timsTOF Pro 2
mass spectrometer operated in the positive mode with TIMS enabled.
A data-dependent acquisition with parallel accumulation-serial fragmentation
(DDA-PASEF) method was utilized. Briefly, a full scan was first acquired
for the mass range of m / z 100 to
1700, with the TIMS 1/ k 0 window set as
0.60–1.60 V·s/cm 2 . For 100% duty cycle (1.17
s cycle time) at a ramp rate of 9.42 Hz, the ramp time and accumulation
time were set to 100 ms, respectively. The precursor isolation window
was set to be linear across the m / z range, with a width of 2 m / z at
700 m / z and 3 m / z at 800 m / z .
Precursors with charge states up to +5 that passed intensity threshold
(2.5 × 10 3 ) were then selected for fragmentation.
Ten PASEF ramps were allowed during each cycle, with a dynamic exclusion
duration of 0.4 min. Nitrogen was used as the collision gas, and the
collision energy ranged from 20 to 59 eV across the defined TIMS 1/ k 0 window. The MS data were searched with
the Bruker Parallel Search Engine
in Real-time (PaSER) platform (ver. 2023) against a reviewed human
protein database from Uniprot (downloaded on 3/30/2023). Enzyme activity
was set to be fully tryptic, with up to two missed cleavages allowed.
The following variable modifications were considered: oxidation on
M (+15.9949 Da) and phosphorylation on S/T/Y (+79.9663 Da), with up
to two modification sites allowed for each peptide. Protein false
discovery rate of 1% was applied. The mass tolerance for both the
precursors and fragments were set to ±20 ppm, with each protein
requiring at least one peptide identified within a mass error of ±10
ppm. Additional postsearch filters were applied to the peptides. XCorr
score cutoff was set to 1.0 for peptides with a charge state of +1
and 0.8 for peptides with charge states of +2 to +4. DeltaCN cutoff
was set to 0.1 for all peptides. A minimum percentage of identified
by ions was set to 40%. Heatmap Generation PaSER data in
an Excel spreadsheet
were filtered to remove immunoglobulin, keratin, and trypsin identifications.
The spectral count values were converted to log 2 values.
These data were further filtered to remove any protein entry with
log 2 values less than 1 across all samples analyzed. The
resultant spreadsheet was passed to R Studio and plotted using the
Heatmap function and the ComplexHeatmap, circlize, and ggplot2 libraries. IP-to-MS Analysis of Four Characterized Patient Sera Sera
antibodies were bound to PrismA resin. Ten microliters PrismA
resin was washed twice with 300 μL IPP buffer in RC tubes. The
RC tubes were sealed, then 50 μL IPP buffer was added to the
PrismA resin. Next, 20 μL serum was added to each tube, followed
by a 45 min incubation at 4 °C with rotation. After this incubation,
the resin was washed four times with 300 μL PBS, then two times
with 200 μL IPP buffer, and the RC tubes were sealed. During binding of antibodies to PrismA resin, K562 lysate was brought
to a concentration of 1 mg/mL in IP-LB. Prior to optimization of ProMTag
coupling, we used an irreversible version of the ProMTag, called PerMTag,
which couples to proteins via an NHS ester. PerMTag was added to the
lysate at a ratio of 1.8 μg of PerMTag per microgram of protein,
and the labeling reaction was incubated for 30 min on ice. The labeling
reaction was quenched by addition of 30 μL quencher per 200
μg lysate followed by a 30 min incubation on ice. The
PerMTagged K562 lysate (200 μg per sample) was then added
to the prepared PrismA resin. The samples were incubated at 4 °C
for 2 h with rotation. The resin was then washed four times with 300
μL IPP buffer. To elute proteins from the PrismA resin, 25 μL
IP-EB was added to the resin followed by a 10 min incubation at room
temperature with rotation. This elution step was repeated once with
both eluates for each sample being collected in the same 1.5 mL tube. The eluates from the PrismA resin were then added to 40 μL
TCO resin that had been washed with 300 μL IP-WB3 in an RC tube.
The samples were incubated for 15 min at room temperature with rotation.
The TCO resin was then washed as follows: one time with 300 μL
IP-WB1, two times with 300 μL IP-WB2, one time with 300 μL
IP-WB3, and two times with 300 μL ultrapure water. Next,
40 μL 100 mM FA was added to the TCO resin followed
by 20 μL MT-Trypsin. Samples were incubated at 37 °C for
1 h. The eluate was collected by brief centrifugation of the RC tube,
then an additional 40 μL 100 mM FA was added to the TCO resin.
Samples were incubated at room temperature with rotation for 15 min.
The RC tubes were once again briefly centrifuged into the same 1.5
mL tube as for the previous eluate. The eluates were then dried fully
in a SpeedVac and stored at −80 °C. Tryptic peptides
were separated by reverse phase XSelect CSH C18
2.5 μm resin (Waters) on an in-line 150 × 0.075 mm column
using an UltiMate 3000 RSLCnano system (Thermo Fisher Scientific).
Peptides were eluted using a 60 min gradient from 98:2 to 65:35 buffer
A/B ratio (buffer A = 0.1% FA, 0.5% ACN, buffer B = 0.1% FA, 99.9%
ACN). Eluted peptides were ionized by electrospray (2.2 kV) followed
by mass spectrometric analysis on an Orbitrap Exploris 480 mass spectrometer
(Thermo Fisher Scientific). To assemble a chromatogram library, six
gas-phase fractions were acquired on the Orbitrap Exploris with 4 m / z DIA spectra (4 m / z precursor isolation windows at 30,000 resolution, normalized
AGC target 100%, maximum inject time 66 ms) using a staggered window
pattern from narrow mass ranges using optimized window placements.
Precursor spectra were acquired after each DIA duty cycle, spanning
the m / z range of the gas-phase fraction
(i.e., 496–602 m / z , 60,000
resolution, normalized AGC target 100%, maximum injection time 50
ms). For wide-window acquisitions, the Orbitrap Exploris was configured
to acquire a precursor scan (385–1015 m / z , 60,000 resolution, normalized AGC target 100%, maximum
injection time 50 ms) followed by 50 × 12 m / z DIA spectra (12 m / z precursor
isolation windows at 15,000 resolution, normalized AGC target 100%,
maximum injection time 33 ms) using a staggered window pattern with
optimized window placements. Precursor spectra were acquired after
each DIA duty cycle. Following data acquisition, data were searched
using an empirically
corrected library and a quantitative analysis was performed to obtain
a comprehensive proteomic profile. Proteins were identified and quantified
using EncyclopeDIA and visualized with
Scaffold DIA using 1% false discovery thresholds at both the protein
and peptide level. Protein MS 2 exclusive intensity values
were assessed for quality using ProteiNorm, a tool for systematic
evaluation of normalization methods, imputation of missing values
and comparisons of multiple differential abundance methods. Normalization methods evaluated included log 2 normalization (log 2 ), median normalization (median),
mean normalization (mean), variance stabilizing normalization (VSN), quantile normalization (quantile), cyclic loess normalization (cyclic loess), global robust linear regression normalization, and global intensity normalization (global intensity).
The individual performance of each method was evaluated by comparing
of the following metrices: total intensity, pooled intragroup coefficient
of variation, pooled intragroup median absolute deviation, pooled
intragroup estimate of variance, intragroup correlation, sample correlation
heatmap (Pearson), and log 2 -ratio distributions. The VSN
normalized data were used to perform statistical analysis using linear
models for microarray data (limma) with empirical Bayes (eBayes) smoothing
to the standard errors. Proteins with
an FDR adjusted p -value < 0.05 and a fold change
> 2 were considered significant. IP of Standard Set without
ProMTag Sera antibodies
or anti-Hsp90 were bound to rProtein A resin. Twenty microliters rProtein
A resin was washed twice with 200 μL IPP buffer in RC tubes.
The RC tubes were sealed, then 30 μL IPP buffer was added to
the rProtein A resin. Next, either 20 μL serum or 5 μL
anti-Hsp90 was added to each tube, followed by a 45 min incubation
at 4 °C with rotation. After this incubation, the resin was washed
three times with 200 μL IPP buffer, then resuspended in 50 μL
IPP buffer. Antibodies were labeled with CyDye Cy5-NHS minimal dye
(Cytiva; 14 μM) for 15 min at 4 °C with rotation. The labeling
reaction was quenched by addition of 2 μL quencher followed
by a 30 min incubation at 4 °C with rotation. The resin was then
washed three times with 200 μL IPP and the RC tubes were sealed. During binding of antibodies to rProtein A resin, 1260 μg
K562 lysate was brought to a concentration of 0.5 mg/mL in IP-LB.
Proteins were labeled with CyDye Cy3-NHS minimal dye (Cytiva; 5.9
μM) for 30 min on ice. The labeling reaction was quenched by
addition of Quencher followed by a 30 min incubation on ice. The Cy3-K562 lysate (200 μg) was then added to the Cy5-antibody
rProtein A resin. The samples were incubated at 4 °C for 2 h
with rotation. The resin was then washed three times with 200 μL
IPP buffer. To elute proteins from the rProtein A resin, 25 μL
IP-EB was added to the resin followed by a 10 min incubation at room
temperature with rotation. This elution step was repeated once with
both eluates for each sample being collected in the same 1.5 mL tube. The eluates were run on a 4–20% SDS-PAGE gel (Bio-Rad) for
∼1.5 h at 120 V until the dye front had completely run out
of the gel. Fluorescence images were acquired using a FluorChem M
imager (ProteinSimple). Images were cropped and despeckled using ImageJ. IP of Standard Set without ProMTag, with Cross-Linking Binding
of sera antibodies or anti-Hsp90 to rProtein A resin and
labeling with Cy5-NHS was carried out as described above. The following
additional cross-linking steps were carried out. Following the 30
min quenching incubation, the resin was washed once with 200 μL
200 mM triethanolamine (TEA) pH 8.9 (Sigma-Aldrich). Next, 100 μL
50 mM dimethyl pimelimidate (Sigma-Aldrich) in TEA was added and the
samples were incubated at 4 °C for 1 h with mixing. The resin
was washed once with 200 μL 200 mM TEA, then 100 μL 100
mM ethanolamine pH 8.9 (Sigma-Aldrich) was added followed by a 15
min incubation at 4 °C with rotation. The resin was washed once
with 200 μL 100 mM FA and then three times with 200 μL
IPP buffer. Labeling of the K562 lysate, binding of the Cy3-K562
lysate to the cross-linked Cy5-antibody resin, washing of the rProtein
A resin, and elution from the rProtein A resin were carried out as
described above. Eluates were run on a 4–20% SDS-PAGE gel (Bio-Rad)
for ∼1.5 h at 120 V until the dye front had completely run
out of the gel. Fluorescence images were acquired using a FluorChem
M imager. Images were cropped and despeckled using ImageJ. IP of
Standard Set with ProMTag Binding of sera antibodies
or anti-Hsp90 to rProtein A resin and labeling with Cy5-NHS was carried
out as described above. Labeling of the K562 lysate was carried out
as described above with the following modification: immediately after
adding the CyDye Cy3-NHS to the K562 lysate, ProMTag was added at
a ratio of 1.8 μg of ProMTag per microgram of protein. The labeling
reaction was incubated and quenched as described above. Binding of
the Cy3-K562 lysate to the Cy5-antibody resin, washing of the rProtein
A resin, and elution from the rProtein A resin were carried out as
described above. The eluates from the rProtein A resin were
then added to 20 μL TCO resin that had been washed with 200
μL IP-WB3 in a RC tube. The samples were incubated for 15 min
at room temperature with rotation. The TCO resin was then washed as
follows: three times with 200 μL IP-EB, three times with 200
μL IP-WB1, two times with 200 μL IP-WB2, one time with
200 μL IP-WB3, and two times with 200 μL ultrapure water. To elute proteins from the TCO resin, 22 μL 100 mM FA, 1%
SDS was added to the resin followed by a 10 min incubation at room
temperature with rotation. This elution step was repeated once with
both eluates for each sample being collected in the same 1.5 mL tube.
The eluate was neutralized by addition of 7 μL 1 M TEAB. Eluates
were run on a 4–20% SDS-PAGE gel (Bio-Rad) for ∼1.5
h at 120 V until the dye front had completely run out of the gel.
Fluorescence images were acquired using a FluorChem M imager. Images
were cropped and despeckled using ImageJ. MS Identification of Bands
Excised from Standard Sera Set Gels The bands excised from
the SDS-PAGE gels shown in Figure S3 were
rinsed and equilibrated in 0.1 M ammonium bicarbonate.
Proteins were reduced with DTT and alkylated with 2-iodoacetamide
while in the gel. For efficient digestion of proteins, the gel bands
were crushed with a pestle in tight-fitting 1.5 mL tubes, then the
gel was dehydrated with ACN. MT-Trypsin in 0.1 M ammonium bicarbonate
was added to the dehydrated gel, and proteins were digested for 2
h at 37 °C. Peptides were extracted from the gel using FA and
ACN, and MT-Trypsin was removed from the solution via capture on TCO
resin. The final peptide eluate was lyophilized prior to MS. Twenty percent of yielded peptides were separated by an Evosep One
high-performance liquid chromatography system (Evosep) and then subjected
to tandem mass spectrometric analyses by a timsTOF Pro 2 mass spectrometer
(Bruker). Briefly, each Evotip Pure desalting column (EV2011, Evosep)
was conditioned and equilibrated following the protocol established
by the manufacturer with minor changes. Decreased centrifugation times
(10–15 s at 700 g ) were applied to keep the
solvent level within 2 mm above the disks in the tip. Additional washing
steps (2× 20 μL 0.1% FA in water) were performed after
peptide loading. The Evosep 100 samples-per-day (100SPD) method was
applied for peptide separation over an 11.5 min gradient using 0.1%
FA in water and 0.1% FA in ACN (Optima LC/MS grade, Fisher Chemical)
as solvent A and B respectively. For data acquisition, a standard
data-dependent acquisition with
parallel accumulation-serial fragmentation (DDA-PASEF) method was
utilized as described above in the “ ” section. All MS data
were searched with MaxQuant (ver. 2.1.3.0, Max Planck
Institute of Biochemistry) against a
reviewed Swiss-Prot human protein sequence database (downloaded on
01/22/2023). For peptide identification, the following modifications
were considered: carbamidomethyl on cysteine (static), oxidation on
methionine (variable), and acetylation of protein N-terminus (variable),
with up to two variable modifications allowed for each peptide. Mass
tolerance was set at ±10 pm, and both unique and razor peptides
were used for protein quantification. Default score and delta score
cutoffs were applied for peptide filtering, and 1% false discovery
rate (FDR) was applied on both peptide-spectrum-match and protein
levels. Cohort Development for Comparison of IP-to-MS to Conventional
IP, Double Immunodiffusion, and ELISA Sera derived from a
subset of SSc patients from the University of Pittsburgh longitudinal
cohort previously classified as ATA+ by IP were assessed by a combined
ELISA and DID testing process using recombinant human topoisomerase
I as substrate antigen and known ATA+ sera as reference samples. A
subset of these sera samples was further analyzed by IP-to-MS. ELISA
of SSc Patient Sera IgG anti-full-length human
topoisomerase I antibody levels were measured using standard solid-phase
ELISA according to the following protocol. Ninety-six well microtiter
plates (Thermo Fisher Scientific) were coated with baculovirus-expressed,
full-length human topoisomerase I purified from Sf9 insect cells (1.0
μg/mL) versus no antigen in carbonate
buffer (50 mM NaHCO 3 /Na 2 CO 3 , pH 9.6)
and incubated overnight at 4 °C. The plates were washed three
times with PBS containing 0.05% Tween-20. Wells were blocked with
PBS-0.05% Tween-20 containing 1% BSA, then appropriately diluted serum
samples (1:10,000) were added for 2 h. The plates were washed and
then incubated for 60 min with horseradish peroxidase-conjugated goat
antihuman IgG (1:10,000; Abcam, Massachusetts, USA). Enzymatic reactions
were initiated using 3,3′,5,5′-tetramethyl-benzidine
(TMB) (Sigma-Aldrich) and then terminated with 1 N H 2 SO 4 . Color development was measured at 450 nm with a BioTek Synergy
2 ELISA Reader (BioTek Company) and quantified as standard units following
conversion of adjusted OD 450 values [OD 450 substrate
antigen – OD 450 no antigen] using a standard reference
serum and dose response curve. All assays were performed in duplicate
wells. Ouchterlony DID of SSc Patient Sera Sixty mm Petri
dishes were used to prepare 0.8% agarose gel [0.8% w/v ultrapure agarose
(Thermo Fisher Scientific) diluted in PBS] templates for Ouchterlony
DID assays. Individual wells were loaded with either 16 μL of
recombinant topoisomerase antigen diluted in PBS to a final concentration
of 0.0625–0.25 mg/mL or patient sera at various dilutions (undiluted,
1:4 dilution in PBS) and incubated at room temperature in a humidified
chamber for 48–72 h. Precipitin lines were then visualized
with white light and scored according to line intensity: 1-faint line
with bright light, 2-moderately strong line with bright light, 3-faint
line with ambient light, 4-moderate/strong line with ambient light. IP-to-MS of SSc Patient Sera K562 lysate was brought
to a concentration of 0.5 mg/mL in IP-LB. PerMTag was added to the
lysate at a ratio of 3.33 μg of lysate per microgram of PerMTag.
The labeling reaction was incubated on ice for 30 min, then it was
quenched by addition of Quencher followed by a 30 min incubation on
ice. The labeled lysate was split into four equal aliquots and stored
at −80 °C. To bind sera antibodies to rProtein A
Sepharose Fast Flow resin, 10 μL resin was washed twice with
100 μL IPP buffer in RC tubes. The RC tubes were sealed, then
30 μL IPP buffer was added to suspend the resin. Next, 10 μL
serum was added to each tube, followed by a 45 min incubation at 4
°C with rotation. During this incubation, the PerMTagged K562
lysate that had been stored at −80 °C was thawed on ice.
Following the 45 min incubation, the rProtein A resin was washed four
times with 100 μL IPP buffer, and the RC tubes were resealed. The PerMTagged K562 lysate (100 μg per sample) was added
to the prepared rProtein A resin and the samples were incubated at
4 °C for 2 h with rotation. The resin was washed four times with
100 μL IPP buffer. To elute proteins from the rProtein A resin,
12.5 μL IP elution buffer was added to the resin followed by
a 10 min incubation at room temperature with rotation. This elution
step was repeated once with both eluates for each sample being collected
in the same 1.5 mL tube. To the combined 25 μL IP eluates,
1 μL 100 mM DTT was
added followed by a 30 min incubation at 56 °C. To alkylate the
samples, 1 μL 200 mM IAA was added followed by a 30 min incubation
at room temperature in the dark. The reduced and alkylated IP
eluates were then added to 10 μL
TCO resin that had been washed with 100 μL IP-WB3. The samples
were incubated for 15 min at room temperature with rotation. The TCO
resin was then washed as follows: two times with 100 μL IP-EB,
two times with 100 μL IP-WB1, two times with 100 μL IP-WB2,
one time with 100 μL IP-WB3, and two times with 100 μL
ultrapure water. Next, 20 μL 100 mM FA was added to the
TCO resin followed
by 10 μL MT-Trypsin. Samples were incubated at 37 °C for
1 h, then the eluate was collected by brief centrifugation of the
RC tube. An additional 20 μL 100 mM FA was added to the TCO
resin and the samples were incubated at room temperature with rotation
for 15 min. The RC tubes were briefly centrifuged into the same tube
as for the previous eluate. Peptides were fully dried in a SpeedVac
and stored at −80 °C. MS of peptides was carried
out as described above in the “ ” section
with one modification: in addition to oxidation on M and phosphorylation
on S/T/Y, carbamidomethyl on C was also considered. The heatmap of
the SSc IP-to-MS results was carried out as described above in the
“ ” section. Statistical Analysis of DID, ELISA, and IP-to-MS Results Concordance of ELISA and DID results was assessed through contingency
table analysis. Phenotypic differences between ATA+ and ATA–
groups classified by the combined ELISA + DID method were analyzed
through a combination of parametric and nonparametric statistical
tests, assuming DID to be the gold standard method for ATA detection. 

K562 cells were grown
by Cell Culture Company. We used the erythroleukemoid cell line K562
because these cells grow rapidly in culture and, although not tissue-specific,
express a wide range of proteins that include commonly targeted autoantigens.
As such, these cells represent the gold standard in the field of IP/autoantibody
detection. To prepare K562 cell lysates, cells were suspended
in IP lysis buffer (IP-LB; Impact Proteomics, LLC.). The cells were
sonicated on ice for 30–40 pulses at 30% power and 30% duty
cycle on a Branson 450 Sonifier. The sample was centrifuged at 14,000
rpm for 20 min in a refrigerated benchtop centrifuge at 4 °C.
The supernatant was removed, and the lysate was stored in 500 μL
aliquots at −80 °C. Protein concentration was determined
via a Pierce BCA assay (Thermo Fisher Scientific). Patient-derived
samples utilized in experiments shown in – were collected
as part of two separate longitudinal
registries at the University of Pittsburgh encompassed by IRB-approved
protocols 20030223 and 19090054. All patients (excluding healthy controls)
had interstitial lung disease associated with an underlying connective
tissue disease such as scleroderma, idiopathic inflammatory myopathy,
or systemic lupus erythematosus. 

Six samples of 50 μg of
K562 lysate were brought to a concentration
of 0.5 mg/mL with IP-LB. Varying amounts of 30 mg/mL ProMTag was added
(0, 0.25, 0.5, 1.0, 3.0, or 5.0 μL); 100% acetonitrile (ACN)
(5.0, 4.75, 4.5, 4.0, 2.0, or 0 μL) was added to bring each
sample to a final volume of 55 μL. Samples were incubated on
ice for 30 min, then 10 μL of Quencher (Impact Proteomics, LLC.)
was added to each sample to quench the labeling reaction. The samples
were incubated on ice for another 30 min. Samples were then incubated
at 4 °C with rotation for 2 h. After the 2 h incubation
was complete, 20% sodium dodecyl sulfate (SDS) (Bio-Rad) was added
to a final concentration of 1.07% and the samples were incubated at
room temperature with rotation for 15 min. During this incubation,
100 μL TCO agarose resin (Vector Laboratories, Inc.) per sample
was washed once with water in resin capture (RC) tubes, which have
a small slit in the bottom that allows the passing of liquid with
minimal dead volume, but retains the solid resin (Impact Proteomics,
LLC.). The samples were added to the TCO resin and incubated at room
temperature with rotation for 15 min. The flowthrough was collected
by brief centrifugation of the RC
tubes. The resin was then washed twice with 200 μL 100 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid) (HEPES), 1% SDS and once with 200 μL 1 mM HEPES, 1% SDS.
Each wash was collected in separate 1.5 mL tubes. To elute the
protein from the TCO resin, 75 μL of 100 mM
formic acid (FA), 1% SDS was added followed by a 10 min incubation
at room temperature with rotation. The elution step was repeated once,
with both eluates being collected into the same 1.5 mL tube by brief
centrifugation of the RC tubes. For each sample, the flowthrough
was combined with the first wash.
The flowthrough + wash 1 fraction and the eluate were dried fully
in a SpeedVac. The flowthrough + wash 1 fraction was resuspended in
100 μL water, and the eluate was resuspended in 100 μL
100 mM HEPES pH 8.0. The samples were run on 4–20% SDS-polyacrylamide
gel electrophoresis (PAGE) gels (Bio-Rad) for 1.5 h at 120 V and the
gels were stained with Coomassie Brilliant Blue R-250 (Bio-Rad). Quantification
of the SDS-PAGE gels was done with ImageJ, with the blank region between
the third and fourth lanes used for background subtraction. The percent
of protein remaining in the flowthrough + wash 1 or the percent of
protein in the eluate was quantified according to the pixel intensity
of the gel images using ImageJ. The protein amount was calculated
as a percentage of the “0 ProMTag” control for the “flowthrough
+ wash 1” gel or as a percentage of the 12.5 μg ProMTag
per μg lysate sample for the “eluate” gel. 

HeLa
lysate in IP-LB was labeled with ProMTag for 30 min at room
temperature at a ratio of 1.67 μg of protein per microgram of
ProMTag. The labeling reaction was quenched by addition of Quencher
followed by a 30 min incubation at room temperature. During
labeling of HeLa lysate, protein A resin was prepared as follows:
5 μL HiTrap MabSelect PrismA resin (Cytiva Life Sciences) was
added to a RC tube and washed 3 times with 300 μL IPP buffer
(Impact Proteomics, LLC.). The RC tube was sealed and the PrismA resin
was resuspended in 25 μL IPP, then 2.5 μL polyclonal anti-Hsp90
(Abcam) and 10 μL patient serum with anti-Topo I autoantibody
was added. Antibody binding to the PrismA resin was carried out for
1.5 h at room temperature with end-over-end rotation. The resin was
then washed 4 times with 300 μL phosphate-buffered saline (PBS)
and 3 times with 300 μL IPP. The resin was suspended in 50 μL
IPP, then CyDye Cy5-NHS minimal dye (Cytiva) was added to a concentration
of 20 μM and labeling was carried out for 15 min at room temperature.
The labeling reaction was quenched by addition of Quencher followed
by a 15 min incubation at room temperature. The resin was washed 2
times with 300 μL IPP and the RC tube was sealed with a stopper
that prevents liquid flow. The ProMTagged HeLa lysate (250 μL)
was then added to the
prepared PrismA resin. The resin was incubated with the lysate for
2 h at room temperature. The resin was then washed 4 times with 300
μL IPP. To elute proteins from the PrismA resin, 50 μL
100 mM HEPES pH 8.0, 1% SDS was added to the resin followed by a 10
min incubation at room temperature. This elution step was repeated
once with both eluates for each sample being collected in the same
1.5 mL tube. One-third of the PrismA resin eluate (33 μL)
was used as
the Load sample. This fraction, which contained Cy5-labeled proteins
that had been bound and eluted from the PrismA beads (mostly Cy5-immunoglobulins),
was labeled with CyDye Cy3-NHS at 20 μM concentration for 15
min at room temperature and then quenched by adding quencher followed
by a 15 min incubation at room temperature. The second third of the
PrismA eluate was added to 20 μL TCO resin that had been washed
twice with 300 μL IP-WB3 in a RC tube. The TCO resin was incubated
at room temperature for 30 min. The protein fraction that did not
bind to the TCO beads was saved as the flowthrough fraction. The flowthrough
fraction, which also contained Cy5-labeled proteins that had been
bound and eluted from the PrismA beads (mostly Cy5-immunoglobulins),
was labeled with CyDye Cy3-NHS at 20 μM concentration for 15
min at room temperature and quenched by the addition of quencher,
as described above. The final third of the PrismA eluate was
added to 20 μL TCO
resin that had been washed twice with 300 μL IP-WB3 in a RC
tube. The TCO resin was washed 3 times with 300 μL 100 mM HEPES
pH 8.0, 1% SDS, 10 mM dl -dithiothreitol (DTT); after the
first addition of this buffer only, the sample was incubated with
mixing for 30 min at room temperature. The TCO resin was then washed
an additional 2 times with 300 μL 100 mM HEPES pH 8.0, then
resuspended in 50 μL 100 mM HEPES pH 8.0. Protein bound to the
TCO resin was labeled with CyDye Cy3-NHS minimal dye (20 μM)
for 15 min at room temperature before being quenched by addition of
quencher followed by a 15 min incubation at room temperature. The
TCO resin was washed once with 300 μL 100 mM HEPES pH 8.0, then
once with 300 μL 1% SDS. To elute protein from the TCO resin,
33 μL 100 mM FA, 1% SDS was added followed by a 30 min incubation
at 37 °C. The Eluate fraction was collected and neutralized by
addition of 1 M tetraethylammonium bromide (TEAB). Samples were
prepared for SDS-PAGE as follows: 33 μL of the
load, flowthrough, and eluate fractions were mixed with 11 μL
4× sample buffer (Bio-Rad). Samples were run on a 4–20%
SDS-PAGE gel (Bio-Rad) for ∼1.5 h at 120 V until the dye front
had completely run out of the gel. Fluorescence images were acquired
using a custom-built imager. 

Sera antibodies were bound
to rProtein A Sepharose Fast Flow resin (Cytiva) ( , step 1B). Twenty microliters
rProtein A resin was washed twice with 200 μL IPP buffer in
RC tubes. The RC tubes were sealed, then 30 μL IPP buffer was
added to the rProtein A resin. Next, 20 μL serum was added to
each tube, followed by a 45 min incubation at 4 °C with rotation.
After this incubation, the resin was washed four times with 300 μL
IPP buffer and the RC tubes were sealed. During binding of antibodies to rProtein A resin,
K562 lysate was
brought to a concentration of 0.5 mg/mL in IP-LB. ProMTag was added
to the lysate at a ratio of 1.8 μg of ProMTag per microgram
of protein, and the labeling reaction was incubated for 30 min on
ice ( , step
1A). The labeling reaction was quenched by addition of 20 μL
quencher per 200 μg lysate followed by a 30 min incubation on
ice. The ProMTagged K562 lysate (200 μg per sample) was
then added
to the prepared rProtein A resin ( , “Bind ProMTagged target proteins
to Ig on protein A resin”). The samples were incubated at 4
°C for 2 h with rotation. The resin was then washed four times
with 300 μL IPP buffer ( , “wash away unbound ProMTagged proteins”).
To elute proteins from the rProtein A resin, 25 μL IP-elution
buffer (IP-EB; Impact Proteomics, LLC.) was added to the resin followed
by a 10 min incubation at room temperature with rotation ( , “elute ProMTagged
target proteins & Ig”). This elution step was repeated
once with both eluates for each sample being collected in the same
1.5 mL tube. The eluates from the rProtein A resin were then
added to 20 μL
TCO resin that had been washed with 300 μL IP-wash buffer 3
(IP-WB3; Impact Proteomics, LLC.) in an RC tube ( , “add TCO resin”).
The samples were incubated for 15 min at room temperature with rotation.
The TCO resin was then washed as follows: three times with 300 μL
IP-EB, three times with 300 μL IP-Wash Buffer 1 (IP-WB1; Impact
Proteomics, LLC.), two times with 300 μL IP-Wash Buffer 2 (IP-WB2;
Impact Proteomics, LLC.), one time with 300 μL IP-WB3, and two
times with 300 μL ultrapure water ( , “wash away unbound Ig”). Next, 40 μL 100 mM FA was added to the TCO resin ( , “release
target proteins”) followed by 20 μL MT-Trypsin (Impact
Proteomics, LLC.). Samples were incubated at 37 °C for 1 h. The
eluate was collected by brief centrifugation of the RC tube, then
an additional 40 μL 100 mM FA was added to the TCO resin. Samples
were incubated at room temperature with rotation for 15 min. The RC
tubes were once again briefly centrifuged into the same 1.5 mL tube
as for the previous eluate ( , “elute target protein peptides ready for MS”).
The eluates were then dried fully in a SpeedVac and stored at −80
°C. 

Each sample was
desalted using an Evotip Pure C18 disposable tip (EV2011, Evosep)
following the manufacturer’s protocol with the following modifications:
(1) for conditioning, instead of soaking the tips in 1-propanol, the
Evotips were washed with 20 μL of 2-propanol (Optima LC/MS grade,
Fisher Chemical) and centrifuged at 700 g for 45–50
s until the solvent level was within 1 mm above the packing material;
(2) the centrifugation steps for rinsing, equilibration, and washing
were shortened to 10–15 s; (3) both the rinse and wash steps
were repeated twice with 20 μL 0.1% FA in water (Optima LC/MS
grade, Fisher Chemical). With an Evosep One HPLC (Evosep), the
desalted peptides were eluted off of the Evotip and loaded on to an
Evosep EV1109 performance analytical column (8 cm × 150 μm
inner diameter, 1.5 μm ReproSil Saphir C18 beads from Dr. Maisch).
Peptide separation was carried out according to the manufacturer’s
preset 11.5 min, 100 samples-per-day (SPD), method with 0.1% FA in
water as solvent A and 0.1% FA in ACN as solvent B (Optima LC/MS grade,
Fisher Chemical). All mass spectrometric data were collected
with a timsTOF Pro 2
mass spectrometer operated in the positive mode with TIMS enabled.
A data-dependent acquisition with parallel accumulation-serial fragmentation
(DDA-PASEF) method was utilized. Briefly, a full scan was first acquired
for the mass range of m / z 100 to
1700, with the TIMS 1/ k 0 window set as
0.60–1.60 V·s/cm 2 . For 100% duty cycle (1.17
s cycle time) at a ramp rate of 9.42 Hz, the ramp time and accumulation
time were set to 100 ms, respectively. The precursor isolation window
was set to be linear across the m / z range, with a width of 2 m / z at
700 m / z and 3 m / z at 800 m / z .
Precursors with charge states up to +5 that passed intensity threshold
(2.5 × 10 3 ) were then selected for fragmentation.
Ten PASEF ramps were allowed during each cycle, with a dynamic exclusion
duration of 0.4 min. Nitrogen was used as the collision gas, and the
collision energy ranged from 20 to 59 eV across the defined TIMS 1/ k 0 window. The MS data were searched with
the Bruker Parallel Search Engine
in Real-time (PaSER) platform (ver. 2023) against a reviewed human
protein database from Uniprot (downloaded on 3/30/2023). Enzyme activity
was set to be fully tryptic, with up to two missed cleavages allowed.
The following variable modifications were considered: oxidation on
M (+15.9949 Da) and phosphorylation on S/T/Y (+79.9663 Da), with up
to two modification sites allowed for each peptide. Protein false
discovery rate of 1% was applied. The mass tolerance for both the
precursors and fragments were set to ±20 ppm, with each protein
requiring at least one peptide identified within a mass error of ±10
ppm. Additional postsearch filters were applied to the peptides. XCorr
score cutoff was set to 1.0 for peptides with a charge state of +1
and 0.8 for peptides with charge states of +2 to +4. DeltaCN cutoff
was set to 0.1 for all peptides. A minimum percentage of identified
by ions was set to 40%. 

PaSER data in
an Excel spreadsheet
were filtered to remove immunoglobulin, keratin, and trypsin identifications.
The spectral count values were converted to log 2 values.
These data were further filtered to remove any protein entry with
log 2 values less than 1 across all samples analyzed. The
resultant spreadsheet was passed to R Studio and plotted using the
Heatmap function and the ComplexHeatmap, circlize, and ggplot2 libraries. 

Sera
antibodies were bound to PrismA resin. Ten microliters PrismA
resin was washed twice with 300 μL IPP buffer in RC tubes. The
RC tubes were sealed, then 50 μL IPP buffer was added to the
PrismA resin. Next, 20 μL serum was added to each tube, followed
by a 45 min incubation at 4 °C with rotation. After this incubation,
the resin was washed four times with 300 μL PBS, then two times
with 200 μL IPP buffer, and the RC tubes were sealed. During binding of antibodies to PrismA resin, K562 lysate was brought
to a concentration of 1 mg/mL in IP-LB. Prior to optimization of ProMTag
coupling, we used an irreversible version of the ProMTag, called PerMTag,
which couples to proteins via an NHS ester. PerMTag was added to the
lysate at a ratio of 1.8 μg of PerMTag per microgram of protein,
and the labeling reaction was incubated for 30 min on ice. The labeling
reaction was quenched by addition of 30 μL quencher per 200
μg lysate followed by a 30 min incubation on ice. The
PerMTagged K562 lysate (200 μg per sample) was then added
to the prepared PrismA resin. The samples were incubated at 4 °C
for 2 h with rotation. The resin was then washed four times with 300
μL IPP buffer. To elute proteins from the PrismA resin, 25 μL
IP-EB was added to the resin followed by a 10 min incubation at room
temperature with rotation. This elution step was repeated once with
both eluates for each sample being collected in the same 1.5 mL tube. The eluates from the PrismA resin were then added to 40 μL
TCO resin that had been washed with 300 μL IP-WB3 in an RC tube.
The samples were incubated for 15 min at room temperature with rotation.
The TCO resin was then washed as follows: one time with 300 μL
IP-WB1, two times with 300 μL IP-WB2, one time with 300 μL
IP-WB3, and two times with 300 μL ultrapure water. Next,
40 μL 100 mM FA was added to the TCO resin followed
by 20 μL MT-Trypsin. Samples were incubated at 37 °C for
1 h. The eluate was collected by brief centrifugation of the RC tube,
then an additional 40 μL 100 mM FA was added to the TCO resin.
Samples were incubated at room temperature with rotation for 15 min.
The RC tubes were once again briefly centrifuged into the same 1.5
mL tube as for the previous eluate. The eluates were then dried fully
in a SpeedVac and stored at −80 °C. Tryptic peptides
were separated by reverse phase XSelect CSH C18
2.5 μm resin (Waters) on an in-line 150 × 0.075 mm column
using an UltiMate 3000 RSLCnano system (Thermo Fisher Scientific).
Peptides were eluted using a 60 min gradient from 98:2 to 65:35 buffer
A/B ratio (buffer A = 0.1% FA, 0.5% ACN, buffer B = 0.1% FA, 99.9%
ACN). Eluted peptides were ionized by electrospray (2.2 kV) followed
by mass spectrometric analysis on an Orbitrap Exploris 480 mass spectrometer
(Thermo Fisher Scientific). To assemble a chromatogram library, six
gas-phase fractions were acquired on the Orbitrap Exploris with 4 m / z DIA spectra (4 m / z precursor isolation windows at 30,000 resolution, normalized
AGC target 100%, maximum inject time 66 ms) using a staggered window
pattern from narrow mass ranges using optimized window placements.
Precursor spectra were acquired after each DIA duty cycle, spanning
the m / z range of the gas-phase fraction
(i.e., 496–602 m / z , 60,000
resolution, normalized AGC target 100%, maximum injection time 50
ms). For wide-window acquisitions, the Orbitrap Exploris was configured
to acquire a precursor scan (385–1015 m / z , 60,000 resolution, normalized AGC target 100%, maximum
injection time 50 ms) followed by 50 × 12 m / z DIA spectra (12 m / z precursor
isolation windows at 15,000 resolution, normalized AGC target 100%,
maximum injection time 33 ms) using a staggered window pattern with
optimized window placements. Precursor spectra were acquired after
each DIA duty cycle. Following data acquisition, data were searched
using an empirically
corrected library and a quantitative analysis was performed to obtain
a comprehensive proteomic profile. Proteins were identified and quantified
using EncyclopeDIA and visualized with
Scaffold DIA using 1% false discovery thresholds at both the protein
and peptide level. Protein MS 2 exclusive intensity values
were assessed for quality using ProteiNorm, a tool for systematic
evaluation of normalization methods, imputation of missing values
and comparisons of multiple differential abundance methods. Normalization methods evaluated included log 2 normalization (log 2 ), median normalization (median),
mean normalization (mean), variance stabilizing normalization (VSN), quantile normalization (quantile), cyclic loess normalization (cyclic loess), global robust linear regression normalization, and global intensity normalization (global intensity).
The individual performance of each method was evaluated by comparing
of the following metrices: total intensity, pooled intragroup coefficient
of variation, pooled intragroup median absolute deviation, pooled
intragroup estimate of variance, intragroup correlation, sample correlation
heatmap (Pearson), and log 2 -ratio distributions. The VSN
normalized data were used to perform statistical analysis using linear
models for microarray data (limma) with empirical Bayes (eBayes) smoothing
to the standard errors. Proteins with
an FDR adjusted p -value < 0.05 and a fold change
> 2 were considered significant. 

Sera antibodies
or anti-Hsp90 were bound to rProtein A resin. Twenty microliters rProtein
A resin was washed twice with 200 μL IPP buffer in RC tubes.
The RC tubes were sealed, then 30 μL IPP buffer was added to
the rProtein A resin. Next, either 20 μL serum or 5 μL
anti-Hsp90 was added to each tube, followed by a 45 min incubation
at 4 °C with rotation. After this incubation, the resin was washed
three times with 200 μL IPP buffer, then resuspended in 50 μL
IPP buffer. Antibodies were labeled with CyDye Cy5-NHS minimal dye
(Cytiva; 14 μM) for 15 min at 4 °C with rotation. The labeling
reaction was quenched by addition of 2 μL quencher followed
by a 30 min incubation at 4 °C with rotation. The resin was then
washed three times with 200 μL IPP and the RC tubes were sealed. During binding of antibodies to rProtein A resin, 1260 μg
K562 lysate was brought to a concentration of 0.5 mg/mL in IP-LB.
Proteins were labeled with CyDye Cy3-NHS minimal dye (Cytiva; 5.9
μM) for 30 min on ice. The labeling reaction was quenched by
addition of Quencher followed by a 30 min incubation on ice. The Cy3-K562 lysate (200 μg) was then added to the Cy5-antibody
rProtein A resin. The samples were incubated at 4 °C for 2 h
with rotation. The resin was then washed three times with 200 μL
IPP buffer. To elute proteins from the rProtein A resin, 25 μL
IP-EB was added to the resin followed by a 10 min incubation at room
temperature with rotation. This elution step was repeated once with
both eluates for each sample being collected in the same 1.5 mL tube. The eluates were run on a 4–20% SDS-PAGE gel (Bio-Rad) for
∼1.5 h at 120 V until the dye front had completely run out
of the gel. Fluorescence images were acquired using a FluorChem M
imager (ProteinSimple). Images were cropped and despeckled using ImageJ. 

Binding
of sera antibodies or anti-Hsp90 to rProtein A resin and
labeling with Cy5-NHS was carried out as described above. The following
additional cross-linking steps were carried out. Following the 30
min quenching incubation, the resin was washed once with 200 μL
200 mM triethanolamine (TEA) pH 8.9 (Sigma-Aldrich). Next, 100 μL
50 mM dimethyl pimelimidate (Sigma-Aldrich) in TEA was added and the
samples were incubated at 4 °C for 1 h with mixing. The resin
was washed once with 200 μL 200 mM TEA, then 100 μL 100
mM ethanolamine pH 8.9 (Sigma-Aldrich) was added followed by a 15
min incubation at 4 °C with rotation. The resin was washed once
with 200 μL 100 mM FA and then three times with 200 μL
IPP buffer. Labeling of the K562 lysate, binding of the Cy3-K562
lysate to the cross-linked Cy5-antibody resin, washing of the rProtein
A resin, and elution from the rProtein A resin were carried out as
described above. Eluates were run on a 4–20% SDS-PAGE gel (Bio-Rad)
for ∼1.5 h at 120 V until the dye front had completely run
out of the gel. Fluorescence images were acquired using a FluorChem
M imager. Images were cropped and despeckled using ImageJ. 

Binding of sera antibodies
or anti-Hsp90 to rProtein A resin and labeling with Cy5-NHS was carried
out as described above. Labeling of the K562 lysate was carried out
as described above with the following modification: immediately after
adding the CyDye Cy3-NHS to the K562 lysate, ProMTag was added at
a ratio of 1.8 μg of ProMTag per microgram of protein. The labeling
reaction was incubated and quenched as described above. Binding of
the Cy3-K562 lysate to the Cy5-antibody resin, washing of the rProtein
A resin, and elution from the rProtein A resin were carried out as
described above. The eluates from the rProtein A resin were
then added to 20 μL TCO resin that had been washed with 200
μL IP-WB3 in a RC tube. The samples were incubated for 15 min
at room temperature with rotation. The TCO resin was then washed as
follows: three times with 200 μL IP-EB, three times with 200
μL IP-WB1, two times with 200 μL IP-WB2, one time with
200 μL IP-WB3, and two times with 200 μL ultrapure water. To elute proteins from the TCO resin, 22 μL 100 mM FA, 1%
SDS was added to the resin followed by a 10 min incubation at room
temperature with rotation. This elution step was repeated once with
both eluates for each sample being collected in the same 1.5 mL tube.
The eluate was neutralized by addition of 7 μL 1 M TEAB. Eluates
were run on a 4–20% SDS-PAGE gel (Bio-Rad) for ∼1.5
h at 120 V until the dye front had completely run out of the gel.
Fluorescence images were acquired using a FluorChem M imager. Images
were cropped and despeckled using ImageJ. 

The bands excised from
the SDS-PAGE gels shown in Figure S3 were
rinsed and equilibrated in 0.1 M ammonium bicarbonate.
Proteins were reduced with DTT and alkylated with 2-iodoacetamide
while in the gel. For efficient digestion of proteins, the gel bands
were crushed with a pestle in tight-fitting 1.5 mL tubes, then the
gel was dehydrated with ACN. MT-Trypsin in 0.1 M ammonium bicarbonate
was added to the dehydrated gel, and proteins were digested for 2
h at 37 °C. Peptides were extracted from the gel using FA and
ACN, and MT-Trypsin was removed from the solution via capture on TCO
resin. The final peptide eluate was lyophilized prior to MS. Twenty percent of yielded peptides were separated by an Evosep One
high-performance liquid chromatography system (Evosep) and then subjected
to tandem mass spectrometric analyses by a timsTOF Pro 2 mass spectrometer
(Bruker). Briefly, each Evotip Pure desalting column (EV2011, Evosep)
was conditioned and equilibrated following the protocol established
by the manufacturer with minor changes. Decreased centrifugation times
(10–15 s at 700 g ) were applied to keep the
solvent level within 2 mm above the disks in the tip. Additional washing
steps (2× 20 μL 0.1% FA in water) were performed after
peptide loading. The Evosep 100 samples-per-day (100SPD) method was
applied for peptide separation over an 11.5 min gradient using 0.1%
FA in water and 0.1% FA in ACN (Optima LC/MS grade, Fisher Chemical)
as solvent A and B respectively. For data acquisition, a standard
data-dependent acquisition with
parallel accumulation-serial fragmentation (DDA-PASEF) method was
utilized as described above in the “ ” section. All MS data
were searched with MaxQuant (ver. 2.1.3.0, Max Planck
Institute of Biochemistry) against a
reviewed Swiss-Prot human protein sequence database (downloaded on
01/22/2023). For peptide identification, the following modifications
were considered: carbamidomethyl on cysteine (static), oxidation on
methionine (variable), and acetylation of protein N-terminus (variable),
with up to two variable modifications allowed for each peptide. Mass
tolerance was set at ±10 pm, and both unique and razor peptides
were used for protein quantification. Default score and delta score
cutoffs were applied for peptide filtering, and 1% false discovery
rate (FDR) was applied on both peptide-spectrum-match and protein
levels. 

Sera derived from a
subset of SSc patients from the University of Pittsburgh longitudinal
cohort previously classified as ATA+ by IP were assessed by a combined
ELISA and DID testing process using recombinant human topoisomerase
I as substrate antigen and known ATA+ sera as reference samples. A
subset of these sera samples was further analyzed by IP-to-MS. 

IgG anti-full-length human
topoisomerase I antibody levels were measured using standard solid-phase
ELISA according to the following protocol. Ninety-six well microtiter
plates (Thermo Fisher Scientific) were coated with baculovirus-expressed,
full-length human topoisomerase I purified from Sf9 insect cells (1.0
μg/mL) versus no antigen in carbonate
buffer (50 mM NaHCO 3 /Na 2 CO 3 , pH 9.6)
and incubated overnight at 4 °C. The plates were washed three
times with PBS containing 0.05% Tween-20. Wells were blocked with
PBS-0.05% Tween-20 containing 1% BSA, then appropriately diluted serum
samples (1:10,000) were added for 2 h. The plates were washed and
then incubated for 60 min with horseradish peroxidase-conjugated goat
antihuman IgG (1:10,000; Abcam, Massachusetts, USA). Enzymatic reactions
were initiated using 3,3′,5,5′-tetramethyl-benzidine
(TMB) (Sigma-Aldrich) and then terminated with 1 N H 2 SO 4 . Color development was measured at 450 nm with a BioTek Synergy
2 ELISA Reader (BioTek Company) and quantified as standard units following
conversion of adjusted OD 450 values [OD 450 substrate
antigen – OD 450 no antigen] using a standard reference
serum and dose response curve. All assays were performed in duplicate
wells. 

Sixty mm Petri
dishes were used to prepare 0.8% agarose gel [0.8% w/v ultrapure agarose
(Thermo Fisher Scientific) diluted in PBS] templates for Ouchterlony
DID assays. Individual wells were loaded with either 16 μL of
recombinant topoisomerase antigen diluted in PBS to a final concentration
of 0.0625–0.25 mg/mL or patient sera at various dilutions (undiluted,
1:4 dilution in PBS) and incubated at room temperature in a humidified
chamber for 48–72 h. Precipitin lines were then visualized
with white light and scored according to line intensity: 1-faint line
with bright light, 2-moderately strong line with bright light, 3-faint
line with ambient light, 4-moderate/strong line with ambient light. 

K562 lysate was brought
to a concentration of 0.5 mg/mL in IP-LB. PerMTag was added to the
lysate at a ratio of 3.33 μg of lysate per microgram of PerMTag.
The labeling reaction was incubated on ice for 30 min, then it was
quenched by addition of Quencher followed by a 30 min incubation on
ice. The labeled lysate was split into four equal aliquots and stored
at −80 °C. To bind sera antibodies to rProtein A
Sepharose Fast Flow resin, 10 μL resin was washed twice with
100 μL IPP buffer in RC tubes. The RC tubes were sealed, then
30 μL IPP buffer was added to suspend the resin. Next, 10 μL
serum was added to each tube, followed by a 45 min incubation at 4
°C with rotation. During this incubation, the PerMTagged K562
lysate that had been stored at −80 °C was thawed on ice.
Following the 45 min incubation, the rProtein A resin was washed four
times with 100 μL IPP buffer, and the RC tubes were resealed. The PerMTagged K562 lysate (100 μg per sample) was added
to the prepared rProtein A resin and the samples were incubated at
4 °C for 2 h with rotation. The resin was washed four times with
100 μL IPP buffer. To elute proteins from the rProtein A resin,
12.5 μL IP elution buffer was added to the resin followed by
a 10 min incubation at room temperature with rotation. This elution
step was repeated once with both eluates for each sample being collected
in the same 1.5 mL tube. To the combined 25 μL IP eluates,
1 μL 100 mM DTT was
added followed by a 30 min incubation at 56 °C. To alkylate the
samples, 1 μL 200 mM IAA was added followed by a 30 min incubation
at room temperature in the dark. The reduced and alkylated IP
eluates were then added to 10 μL
TCO resin that had been washed with 100 μL IP-WB3. The samples
were incubated for 15 min at room temperature with rotation. The TCO
resin was then washed as follows: two times with 100 μL IP-EB,
two times with 100 μL IP-WB1, two times with 100 μL IP-WB2,
one time with 100 μL IP-WB3, and two times with 100 μL
ultrapure water. Next, 20 μL 100 mM FA was added to the
TCO resin followed
by 10 μL MT-Trypsin. Samples were incubated at 37 °C for
1 h, then the eluate was collected by brief centrifugation of the
RC tube. An additional 20 μL 100 mM FA was added to the TCO
resin and the samples were incubated at room temperature with rotation
for 15 min. The RC tubes were briefly centrifuged into the same tube
as for the previous eluate. Peptides were fully dried in a SpeedVac
and stored at −80 °C. MS of peptides was carried
out as described above in the “ ” section
with one modification: in addition to oxidation on M and phosphorylation
on S/T/Y, carbamidomethyl on C was also considered. The heatmap of
the SSc IP-to-MS results was carried out as described above in the
“ ” section. 

Concordance of ELISA and DID results was assessed through contingency
table analysis. Phenotypic differences between ATA+ and ATA–
groups classified by the combined ELISA + DID method were analyzed
through a combination of parametric and nonparametric statistical
tests, assuming DID to be the gold standard method for ATA detection. 

The IP-to-MS Workflow The IP-to-MS workflow is outlined
in . First,
proteins from the desired cell or tissue source are labeled with ProMTag
so that greater than 85% of the input protein is capturable by TCO
resin. Concurrently, patient serum is exposed to protein A resin to
capture immunoglobulins. After washing unbound proteins from the protein
A resin, the ProMTagged whole-cell proteome (autoantigen target pool)
is exposed to patient antibodies bound to the protein A resin. Unbound
ProMTagged proteins are washed away, leaving bound ProMTagged target
proteins and immunoglobulins bound to the protein A resin. All
proteins bound to the protein A resin are then released by SDS denaturation.
This mixture of ProMTagged target proteins and untagged immunoglobulins
is exposed to TCO resin, which covalently cross-links the ProMTagged
target proteins. The TCO resin is then washed extensively to remove
immunoglobulins and any remaining contaminants. At this point
in the workflow, intact target proteins are released
from the TCO resin by a pH change which reverses the linkage between
the ProMTag and the protein, leaving a chemically unaltered protein
ready for further analysis either by gel electrophoresis or trypsin
digestion and MS analysis. To enable rapid trypsin digestion, MT tagged
trypsin (MT-Trypsin) is added in excess to digest the target proteins
within 1 h. MT-Trypsin is removed from solution by covalent coupling
to the TCO resin, thus yielding tryptic peptides ready for MS analysis. A set of control experiments were performed to test for ProMTag
protein capture and release under IP conditions. More than 85% of
the input protein target pool was captured and released ( Figure S1A ). We further demonstrated that the
vast majority of untagged antibodies were separated from ProMTagged
target proteins, thus enabling direct IP of target proteins and efficient
antibody removal ( Figure S1B,C ). IP-to-MS
Analysis of Individuals with Interstitial Lung Disease To
demonstrate the utility of the IP-to-MS workflow, we assessed
32 sera from patients diagnosed with connective tissue disease-associated
interstitial lung disease (CTD-ILD), and five sera from healthy individuals
( and S2 ; Table S1 ). Fifteen of the CTD-ILD sera were previously characterized
and known to recognize proteins including topoisomerase I, exosome
complex, RNase P complex, ribonuclear protein (RNP) complex, RNA polymerase,
X-ray repair complex, centromere, and alanine aminoacyl tRNA synthetase.
These were used as positive controls. The remaining 17 CTD-ILD patient
serum samples had not been fully characterized and therefore had autoantibodies
of unknown specificity. All of these samples were processed by the
IP-to-MS workflow. MS analysis was performed on a Bruker timsTOF Pro2
instrument using an 11.5 min gradient routine. Because the IP-to-MS
samples were known to have relatively low complexity, the shorter
MS run time allowed for higher throughput without sacrificing sensitivity.
The data were filtered to remove immunoglobulin, keratin, and trypsin
identifications. The data were then displayed as a heatmap where the
log 2 spectral counts were plotted. Proteins with at least
two spectral counts in at least one sample were included in the heatmap. The top region of the heatmap contains proteins
that were detected
in most or all of the samples, ranging from high spectral counts to
low spectral counts ( Figure S2 ). Because
of the broad distribution across the majority of sera, these proteins
were deemed to be nonspecific binding proteins. Several of these proteins
were seen only in the patient sera, and not in the healthy controls,
suggesting more specific recognition by cognate autoantibodies. Below this region of putative nonspecific binding proteins lies
proteins that are clearly sample-specific. Vertical streaks are an
obvious feature of this lower section of the heatmap. These groups
of proteins that were specifically immunoprecipitated by individual
patient sera often correspond to subunits of particular protein complexes,
such as ribonuclease P, exosome, RNA polymerases, and RNP complexes
( A–C).
Because the ProMTag is added under native conditions, the IP-to-MS
method immunoprecipitates protein complexes. With respect to
the previously characterized sera that were used
as positive controls, the IP-to-MS method positively identified expected
proteins in 14 out of 15 samples. For example, the three known sera
with topoisomerase I reactivity (Topo.I.1 through Topo.I.3) detected
this protein in the IP-to-MS workflow ( A). In addition, topoisomerase I was also
detected in one of the uncharacterized patient serum samples (Test.1).
The Ku.1 patient serum containing X-ray repair cross-complementing
proteins 5 and 6 antibodies immunoprecipitated corresponding antigen
targets in the IP-to-MS workflow. These proteins were also identified
in the Topo.I.2, Th.To.1, and RNAP.2 patient sera, which were also
reactive against topoisomerase I, ribonuclease P and annexin 11, and
RNA polymerases, respectively ( A,B). All 17 test samples reacted to specific
sets of proteins. Many
of the test samples targeted proteins that were also recognized by
some of the sera from the positive control group, including topoisomerase
I (Test.1), ribonuclease P subunits (Test.4), exosome subunits (Test.2),
and RNA polymerase subunits (Test.5), among others ( A,B). However, the remaining
Test samples appeared to contain antibodies to previously uncharacterized
autoantigen proteins. Of note is annexin A11, which was immunoprecipitated
by Test.3 serum, as well as by positive control sera that had reactivity
to the exosome and ribonuclease P ( A). Other interesting autoantigens were observed,
including the THO complex ( D, Test.9), eukaryotic translation initiation factor
2B (eIF-2B) ( E, Test.7 and Test.17), and phenylalanine tRNA ligase ( F, Test.14). Several proteins
were immunoprecipitated by many of the patient sera, but not by the
healthy control sera. For example, 11 of the 32 patient sera immunoprecipitated
Ro52/TRIM21 (tripartite motif-containing protein 21) ( G), a known autoantigen in
Sjögren’s syndrome, systemic lupus erythematosus, and
other rheumatic autoimmune diseases. − This test of the IP-to-MS workflow demonstrated that IP-to-MS
can
correctly identify autoantigen targets of previously characterized
sera from patients with autoimmune disease. It also showed that all
test sera contained autoantibodies to previously known autoantigens
and/or novel autoantigens, demonstrating the potential utility of
this method for discovering autoantigen targets recognized by patient
sera. IP-to-MS Reproducibility To demonstrate the reproducibility
of the IP-to-MS workflow, four patient sera with known autoantibodies
and a healthy control serum were assessed in triplicate. Each IP-to-MS
sample was analyzed by a 1 h gradient on an Orbitrap Exploris 480
MS. shows
volcano plots comparing each patient serum to control serum taken
from a healthy individual where the average log 2 fold change
(log FC) is plotted against the average −log 10 FDR
adjusted probability (neglog(adj. P)) score for proteins commonly
detected across samples. We took a very stringent approach and focused
on proteins with log 2 FC > ± 1 and −log 10 adj. P > 1.3. Reproducibility was quantified
using the Adjusted P -value which is calculated from
the confidence values, standard error, and corrected for the False
Discovery Rate according to Benjamini and Hochberg. These data are contained in the Table S6 excel spreadsheet, where a total of 2676 proteins were identified
across all 15 samples. Patients 1 and 3 (P1 and P3) have myositis, which
is charactered
by presence of antiaminoacyl tRNA synthetase antibodies. P1 serum contained antibodies that immunoprecipitated
glycine aminoacyl tRNA synthetase (GARS) ( A). Two other interesting proteins were detected
in this P1 versus control IP experiment, including AFG3L2, an ATP-dependent
protease localized to the mitochondrial inner membrane. Curiously,
the control serum immunoprecipitated KIFC1, a mitotic kinesin molecule.
MS analysis of the P3 IP products showed a very strong IP of alanine
aminoacyl tRNA synthetase (AARS), without other significant autoantigens
( C). P2 and P4 sera were obtained from patients with scleroderma with
known anti-topoisomerase I antibodies. − MS analysis of the P2 serum immunoprecipitate showed
the presence of both cytoplasmic topoisomerase I (TOP1) and mitochondrial
topoisomerase 1 (TOP1MT), two highly homologous proteins ( B). Serum P4 immunoprecipitated
the most complex set of autoantigens ( D). In addition to expected TOP1 and TOP1MT,
three other proteins were immunoprecipitated, DLAT (dihydrolipoyllysine-residue
acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial),
CENPC (centromere protein C), and PDHX (pyruvate dehydrogenase protein
X component, mitochondrial) ( D). The normalized intensities corresponding
to specific autoantigen
targets found were plotted for each IP sample . These data demonstrate the high degree
of reproducibility of this method with a tight clustering of each
protein within each IP group. The associated FDR adjusted P -value for each protein is shown in parentheses. Thus,
the IP-to-MS workflow was able to detect both known autoantigens and
novel autoantigens in a highly reproducible manner. Comparison
of IP-to-MS to Other IP Methods To further
demonstrate the utility of the IP-to-MS workflow, we compared our
IP-to-MS method to conventional IP and IP with immunoglobulins chemically
cross-linked to protein A resin. In all three experiments, target/substrate
proteins derived from K562 cell lysate were labeled with Cy3-NHS.
Serum proteins (including immunoglobulins) bound to protein A resin
were Cy5-labeled in situ. Four sera from either myositis or scleroderma
patients with autoantibodies of known specificity were assessed. Included
in this experiment were a negative control serum from a healthy/nondiseased
patient and a positive control preparation of purified rabbit polyclonal
antibodies targeting Hsp90. First, we performed conventional
IPs without ProMTag or chemical cross-linking ( A). The Cy3-labeled target proteins are shown in green, while
the Cy5-labeled immunoglobulins are shown in red. The negative control
lane shows a set of Cy3-labeled proteins from the target cell lysate
that were nonspecifically retained by the workflow. This same set
of proteins was observed across all patient sera lanes, but not in
the purified rabbit antibody lane, indicating that the presence of
either bound immunoglobulins or other serum associated proteins aided
in the nonspecific binding of target cell lysate. In addition to these
so-called background bands, the patient sera precipitated additional
distinct protein bands. The Cy5 fluorescence image also revealed a
variable amount of immunoglobulin associated with each serum sample. A commonly used method for separating
immunoglobulins
from antigens
during IP is to chemically cross-link immunoglobulins to the protein
A resin. Chemical cross-linking has certain limitations. , − If the extent of cross-linking is low, then immunoglobulin
will contaminate the final IP product. Conversely, if the cross-linking
is too aggressive, there is potential to block the antigen binding
sites, reducing the efficacy of IP. Thus, typical IP experiments involving
chemical cross-linking require optimization of the extent of cross-linking
reagent. Here, we used the recommended conditions provided by the
manufacturer, without optimization for each serum tested. Cy3 fluorescence
demonstrated that cross-linking improved the resolution of the IP
gels ( B). However,
the background bands persisted. Furthermore, Cy5 fluorescence showed
that, while the level of immunoglobulin contamination was reduced
compared to the non-cross-linked samples, the variable degree of immunoglobulin
removal indicated insufficient cross-linking in some samples. Using double-labeled Cy3, ProMTagged cell lysate in the IP workflow
showed that the level of background Cy3-labeled proteins was reduced
relative to the Cy3-target proteins ( C, note the relative intensity of the target
protein bands to the background bands seen in the control lane is
stronger). This reduction in background signal was most likely due
to the more stringent washing of the TCO resin afforded by the ProMTag
covalent linkage, though it is important to point out that there was
still some immunoglobulin breakthrough in the final IP product. Overall,
this breakthrough appeared to be less significant than the immunoglobulin
contamination observed in the cross-linking experiment (compare B,C merged images). Finally, to confirm the identity of the Cy3-target protein bands
in these gels, the bands were excised from their respective gels and
the proteins were rapidly digested with MT-Trypsin and analyzed by
MS ( Figure S3 ). Shown are the top ten proteins
identified by MS for each of the 21 protein bands excised from the
three IP gels where there was no treatment of the antibodies bound
to the protein A resin or ProMTagging of the K562 lysate ( Figure S3A ); the antibodies were cross-linked
to the protein A resin ( Figure S3B ); or,
the K562 lysate was labeled with ProMTag ( Figure S3C ). Sera labeled P1 and P3 derived from myositis patients
and were
known to contain antibodies against glycine tRNA synthetase and alanine
tRNA synthetase, respectively. Bands
2, 9, and 16, which were cut from P1 lanes, were confirmed to contain
glycine tRNA synthetase, while bands 4, 11, and 18, which were cut
from P3 lanes, were confirmed to contain alanine tRNA synthetase,
as expected ( Figure S3 ). The P2 and P4
sera were obtained from patients with scleroderma and were known to
contain anti-topoisomerase I antibodies. − Bands 3, 6, 10, 13, 17, and 20, which were cut from
the P2 and P4 lanes, were all confirmed to contain topoisomerase I
as expected. These data clearly confirmed that the various IP methods
isolated the expected proteins, but importantly the ProMTagged products
appeared to be the most contaminant- and immunoglobulin-free. Comparison
of IP-to-MS to Conventional IP, Double Immunodiffusion,
and ELISA Systemic sclerosis (SSc) encompasses a group of
autoimmune diseases characterized by tissue fibrosis, vascular disease,
and autoantibody production. The most prevalent SSc-related autoantibodies
include anti-centromere, anti-topoisomerase I (ATA), and anti-RNA
polymerase III. Importantly, these autoantibodies mark distinct subsets
with varied clinical phenotypes and disease outcomes/prognoses. As
an example of these phenotype–serotype associations, ILD is
the most common cause of death in SSc patients and is more prevalent
among patients positive for ATA, demonstrating that ATA is an indicator
of SSc prognosis. Accurate autoantibody
identification is therefore crucial for optimal clinical management. Currently, IP is the gold standard method for identifying most
SSc-specific autoantibodies, but the reliability of this assay in
determining the presence of ATA is questionable given that topoisomerase
I appears at the same band size as multiple other human proteins/target
antigens. Ouchterlony DID represents an alternative method of detecting
ATA in SSc patient sera, but there has been little research on its
comparative accuracy and reliability relative to IP or other highly
sensitive, but potentially less specific, commercial methods such
as ELISA. To address these questions, we compared the accuracy
of conventional
IP, ELISA, DID, and IP-to-MS in the detection of ATA in SSc patients.
Sera derived from a group of SSc patients from the University of Pittsburgh
longitudinal cohort previously classified as ATA positive (ATA+) by
conventional IP, DID* (calf thymus extract used as substrate), or
commercial ELISA were reassessed by custom ELISA and DID assays using
recombinant human topoisomerase I as substrate antigen. We assessed
82 serum samples through our custom ELISA. As shown
in Table S2 , 66/82 samples exceeded the
threshold of 0.15 arbitrary units (AU), which was established based
on the mean standardized antibody level (+2 SD) in a healthy control
cohort. Based on the distribution of ELISA values across the full
cohort, we further stratified patients into three groups: high titer
(≥30th percentile rank of cohort), low titer (<30th percentile),
and negative (<0.15 AU). When assessed by our custom Ouchterlony
DID assay, 63/82 samples
yielded precipitin lines consistent with the presence of ATA ( Table S2 ). Five of the 19 samples that were negative
by our custom DID were low titer positive ATA by our ELISA. Of the
66 samples with ELISA evidence of ATA, 61 generated DID patterns indicative
of ATA positivity, and 61/63 samples positive for ATA by DID were
also positive by ELISA ( Table S3 ). The
overall concordance of ELISA and DID was 91%. Based on the assumption
that DID represents the conventional “gold standard”
for accurate detection of ATA, the relative sensitivity and specificity
of our custom ELISA was 0.968 and 0.667, respectively. Because 18%
(15/82) of the cohort previously classified as ATA+ by conventional
IP was classified as ATA negative (ATA−) by DID, our results
suggested that conventional IP alone is unreliable in accurately identifying
ATA positivity. To address this apparent discrepancy, we selected
13 SSc+ patient
sera with discordant ATA assessments (ATA+ by conventional IP, DID*,
or commercial ELISA, but ATA– by our custom DID) for analysis
by IP-to-MS ( , P1–P8, P10, P12, P13, P15, and P16; and Table S2 ). Six sera from SSc+ patients that were deemed ATA–
by conventional IP or commercial ELISA were included as an SSc+ ATA–
control group ( , P9, P11, P14, and P17–P19). In addition, seven ATA+ sera
(positive by conventional IP, as well as our custom ELISA and DID
assays) were selected as reference samples ( , P20–P26). Sera from six healthy
individuals comprised the negative control group ( , P27–P32). With IP-to-MS, quantifiable
topoisomerase I was detected
in 13
samples ( , A) based on spectral
counts of unique topoisomerase I peptides. All of the reference samples
(7/7) were high titer. Six of the ATA– (by custom DID) samples
(6/19) also had detectable topoisomerase I by IP-to-MS; five were
strongly positive and one was weakly positive. Topoisomerase I was
undetectable in 13 of the test samples (13/19), all of which were
also ATA– by both ELISA and DID. Importantly, none of the healthy
control sera contained topoisomerase I reactivity. Overall, there was very good concordance between
the four immunoassays
among the reference samples, with the exception of P20 that demonstrated
low titer positivity by ELISA, but high titer positivity based on
IP-to-MS spectral counts. Among the test group, 5 samples demonstrating
low titer positive ATA by ELISA yielded high-titer positive results
by IP-to-MS. One sample (P6) was negative by ELISA and DID but tested
positive by IP-to-MS. Collectively, these results indicate that IP-to-MS
may be a more sensitive measure of anti-topoisomerase I antibodies
than conventional IP, DID, or ELISA. IP-to-MS provides a survey
of the target cell lysate’s whole
proteome. Figure S4 shows a heatmap of
proteins captured by the IP-to-MS workflow for the 32 sera examined.
As in Figure S2 , there were nonspecific
binding proteins detected in the reference, test, and healthy control
sera. Topoisomerase I was not the only protein specifically precipitated
by the reference and test sera. These other proteins included argonaute
subunits, TRIM21, DDX18, and dihydrolipoyllysine acetyltransferase
( B), demonstrating
that IP-to-MS can also serve as an autoantigen discovery tool. As
further evidence of this diagnostic utility, the test sera that were
topoisomerase I negative contained antibodies to novel antigens, including
coilin, nibrin, MRE11, Nup50, elongator proteins, RNA polymerases,
and exosome subunits ( C). Taken together, these data show that IP-to-MS may
outperform conventional
IP, DID, and ELISA in identifying sera that contain ATA. Equally important,
IP-to-MS has the capacity to identify additional, potentially novel/undiscovered
autoantigens recognized by patient sera, allowing for further stratification
of patients than is possible with conventional methods of autoantibody
detection. 

The IP-to-MS workflow is outlined
in . First,
proteins from the desired cell or tissue source are labeled with ProMTag
so that greater than 85% of the input protein is capturable by TCO
resin. Concurrently, patient serum is exposed to protein A resin to
capture immunoglobulins. After washing unbound proteins from the protein
A resin, the ProMTagged whole-cell proteome (autoantigen target pool)
is exposed to patient antibodies bound to the protein A resin. Unbound
ProMTagged proteins are washed away, leaving bound ProMTagged target
proteins and immunoglobulins bound to the protein A resin. All
proteins bound to the protein A resin are then released by SDS denaturation.
This mixture of ProMTagged target proteins and untagged immunoglobulins
is exposed to TCO resin, which covalently cross-links the ProMTagged
target proteins. The TCO resin is then washed extensively to remove
immunoglobulins and any remaining contaminants. At this point
in the workflow, intact target proteins are released
from the TCO resin by a pH change which reverses the linkage between
the ProMTag and the protein, leaving a chemically unaltered protein
ready for further analysis either by gel electrophoresis or trypsin
digestion and MS analysis. To enable rapid trypsin digestion, MT tagged
trypsin (MT-Trypsin) is added in excess to digest the target proteins
within 1 h. MT-Trypsin is removed from solution by covalent coupling
to the TCO resin, thus yielding tryptic peptides ready for MS analysis. A set of control experiments were performed to test for ProMTag
protein capture and release under IP conditions. More than 85% of
the input protein target pool was captured and released ( Figure S1A ). We further demonstrated that the
vast majority of untagged antibodies were separated from ProMTagged
target proteins, thus enabling direct IP of target proteins and efficient
antibody removal ( Figure S1B,C ). 

To
demonstrate the utility of the IP-to-MS workflow, we assessed
32 sera from patients diagnosed with connective tissue disease-associated
interstitial lung disease (CTD-ILD), and five sera from healthy individuals
( and S2 ; Table S1 ). Fifteen of the CTD-ILD sera were previously characterized
and known to recognize proteins including topoisomerase I, exosome
complex, RNase P complex, ribonuclear protein (RNP) complex, RNA polymerase,
X-ray repair complex, centromere, and alanine aminoacyl tRNA synthetase.
These were used as positive controls. The remaining 17 CTD-ILD patient
serum samples had not been fully characterized and therefore had autoantibodies
of unknown specificity. All of these samples were processed by the
IP-to-MS workflow. MS analysis was performed on a Bruker timsTOF Pro2
instrument using an 11.5 min gradient routine. Because the IP-to-MS
samples were known to have relatively low complexity, the shorter
MS run time allowed for higher throughput without sacrificing sensitivity.
The data were filtered to remove immunoglobulin, keratin, and trypsin
identifications. The data were then displayed as a heatmap where the
log 2 spectral counts were plotted. Proteins with at least
two spectral counts in at least one sample were included in the heatmap. The top region of the heatmap contains proteins
that were detected
in most or all of the samples, ranging from high spectral counts to
low spectral counts ( Figure S2 ). Because
of the broad distribution across the majority of sera, these proteins
were deemed to be nonspecific binding proteins. Several of these proteins
were seen only in the patient sera, and not in the healthy controls,
suggesting more specific recognition by cognate autoantibodies. Below this region of putative nonspecific binding proteins lies
proteins that are clearly sample-specific. Vertical streaks are an
obvious feature of this lower section of the heatmap. These groups
of proteins that were specifically immunoprecipitated by individual
patient sera often correspond to subunits of particular protein complexes,
such as ribonuclease P, exosome, RNA polymerases, and RNP complexes
( A–C).
Because the ProMTag is added under native conditions, the IP-to-MS
method immunoprecipitates protein complexes. With respect to
the previously characterized sera that were used
as positive controls, the IP-to-MS method positively identified expected
proteins in 14 out of 15 samples. For example, the three known sera
with topoisomerase I reactivity (Topo.I.1 through Topo.I.3) detected
this protein in the IP-to-MS workflow ( A). In addition, topoisomerase I was also
detected in one of the uncharacterized patient serum samples (Test.1).
The Ku.1 patient serum containing X-ray repair cross-complementing
proteins 5 and 6 antibodies immunoprecipitated corresponding antigen
targets in the IP-to-MS workflow. These proteins were also identified
in the Topo.I.2, Th.To.1, and RNAP.2 patient sera, which were also
reactive against topoisomerase I, ribonuclease P and annexin 11, and
RNA polymerases, respectively ( A,B). All 17 test samples reacted to specific
sets of proteins. Many
of the test samples targeted proteins that were also recognized by
some of the sera from the positive control group, including topoisomerase
I (Test.1), ribonuclease P subunits (Test.4), exosome subunits (Test.2),
and RNA polymerase subunits (Test.5), among others ( A,B). However, the remaining
Test samples appeared to contain antibodies to previously uncharacterized
autoantigen proteins. Of note is annexin A11, which was immunoprecipitated
by Test.3 serum, as well as by positive control sera that had reactivity
to the exosome and ribonuclease P ( A). Other interesting autoantigens were observed,
including the THO complex ( D, Test.9), eukaryotic translation initiation factor
2B (eIF-2B) ( E, Test.7 and Test.17), and phenylalanine tRNA ligase ( F, Test.14). Several proteins
were immunoprecipitated by many of the patient sera, but not by the
healthy control sera. For example, 11 of the 32 patient sera immunoprecipitated
Ro52/TRIM21 (tripartite motif-containing protein 21) ( G), a known autoantigen in
Sjögren’s syndrome, systemic lupus erythematosus, and
other rheumatic autoimmune diseases. − This test of the IP-to-MS workflow demonstrated that IP-to-MS
can
correctly identify autoantigen targets of previously characterized
sera from patients with autoimmune disease. It also showed that all
test sera contained autoantibodies to previously known autoantigens
and/or novel autoantigens, demonstrating the potential utility of
this method for discovering autoantigen targets recognized by patient
sera. 

To demonstrate the reproducibility
of the IP-to-MS workflow, four patient sera with known autoantibodies
and a healthy control serum were assessed in triplicate. Each IP-to-MS
sample was analyzed by a 1 h gradient on an Orbitrap Exploris 480
MS. shows
volcano plots comparing each patient serum to control serum taken
from a healthy individual where the average log 2 fold change
(log FC) is plotted against the average −log 10 FDR
adjusted probability (neglog(adj. P)) score for proteins commonly
detected across samples. We took a very stringent approach and focused
on proteins with log 2 FC > ± 1 and −log 10 adj. P > 1.3. Reproducibility was quantified
using the Adjusted P -value which is calculated from
the confidence values, standard error, and corrected for the False
Discovery Rate according to Benjamini and Hochberg. These data are contained in the Table S6 excel spreadsheet, where a total of 2676 proteins were identified
across all 15 samples. Patients 1 and 3 (P1 and P3) have myositis, which
is charactered
by presence of antiaminoacyl tRNA synthetase antibodies. P1 serum contained antibodies that immunoprecipitated
glycine aminoacyl tRNA synthetase (GARS) ( A). Two other interesting proteins were detected
in this P1 versus control IP experiment, including AFG3L2, an ATP-dependent
protease localized to the mitochondrial inner membrane. Curiously,
the control serum immunoprecipitated KIFC1, a mitotic kinesin molecule.
MS analysis of the P3 IP products showed a very strong IP of alanine
aminoacyl tRNA synthetase (AARS), without other significant autoantigens
( C). P2 and P4 sera were obtained from patients with scleroderma with
known anti-topoisomerase I antibodies. − MS analysis of the P2 serum immunoprecipitate showed
the presence of both cytoplasmic topoisomerase I (TOP1) and mitochondrial
topoisomerase 1 (TOP1MT), two highly homologous proteins ( B). Serum P4 immunoprecipitated
the most complex set of autoantigens ( D). In addition to expected TOP1 and TOP1MT,
three other proteins were immunoprecipitated, DLAT (dihydrolipoyllysine-residue
acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial),
CENPC (centromere protein C), and PDHX (pyruvate dehydrogenase protein
X component, mitochondrial) ( D). The normalized intensities corresponding
to specific autoantigen
targets found were plotted for each IP sample . These data demonstrate the high degree
of reproducibility of this method with a tight clustering of each
protein within each IP group. The associated FDR adjusted P -value for each protein is shown in parentheses. Thus,
the IP-to-MS workflow was able to detect both known autoantigens and
novel autoantigens in a highly reproducible manner. 

To further
demonstrate the utility of the IP-to-MS workflow, we compared our
IP-to-MS method to conventional IP and IP with immunoglobulins chemically
cross-linked to protein A resin. In all three experiments, target/substrate
proteins derived from K562 cell lysate were labeled with Cy3-NHS.
Serum proteins (including immunoglobulins) bound to protein A resin
were Cy5-labeled in situ. Four sera from either myositis or scleroderma
patients with autoantibodies of known specificity were assessed. Included
in this experiment were a negative control serum from a healthy/nondiseased
patient and a positive control preparation of purified rabbit polyclonal
antibodies targeting Hsp90. First, we performed conventional
IPs without ProMTag or chemical cross-linking ( A). The Cy3-labeled target proteins are shown in green, while
the Cy5-labeled immunoglobulins are shown in red. The negative control
lane shows a set of Cy3-labeled proteins from the target cell lysate
that were nonspecifically retained by the workflow. This same set
of proteins was observed across all patient sera lanes, but not in
the purified rabbit antibody lane, indicating that the presence of
either bound immunoglobulins or other serum associated proteins aided
in the nonspecific binding of target cell lysate. In addition to these
so-called background bands, the patient sera precipitated additional
distinct protein bands. The Cy5 fluorescence image also revealed a
variable amount of immunoglobulin associated with each serum sample. A commonly used method for separating
immunoglobulins
from antigens
during IP is to chemically cross-link immunoglobulins to the protein
A resin. Chemical cross-linking has certain limitations. , − If the extent of cross-linking is low, then immunoglobulin
will contaminate the final IP product. Conversely, if the cross-linking
is too aggressive, there is potential to block the antigen binding
sites, reducing the efficacy of IP. Thus, typical IP experiments involving
chemical cross-linking require optimization of the extent of cross-linking
reagent. Here, we used the recommended conditions provided by the
manufacturer, without optimization for each serum tested. Cy3 fluorescence
demonstrated that cross-linking improved the resolution of the IP
gels ( B). However,
the background bands persisted. Furthermore, Cy5 fluorescence showed
that, while the level of immunoglobulin contamination was reduced
compared to the non-cross-linked samples, the variable degree of immunoglobulin
removal indicated insufficient cross-linking in some samples. Using double-labeled Cy3, ProMTagged cell lysate in the IP workflow
showed that the level of background Cy3-labeled proteins was reduced
relative to the Cy3-target proteins ( C, note the relative intensity of the target
protein bands to the background bands seen in the control lane is
stronger). This reduction in background signal was most likely due
to the more stringent washing of the TCO resin afforded by the ProMTag
covalent linkage, though it is important to point out that there was
still some immunoglobulin breakthrough in the final IP product. Overall,
this breakthrough appeared to be less significant than the immunoglobulin
contamination observed in the cross-linking experiment (compare B,C merged images). Finally, to confirm the identity of the Cy3-target protein bands
in these gels, the bands were excised from their respective gels and
the proteins were rapidly digested with MT-Trypsin and analyzed by
MS ( Figure S3 ). Shown are the top ten proteins
identified by MS for each of the 21 protein bands excised from the
three IP gels where there was no treatment of the antibodies bound
to the protein A resin or ProMTagging of the K562 lysate ( Figure S3A ); the antibodies were cross-linked
to the protein A resin ( Figure S3B ); or,
the K562 lysate was labeled with ProMTag ( Figure S3C ). Sera labeled P1 and P3 derived from myositis patients
and were
known to contain antibodies against glycine tRNA synthetase and alanine
tRNA synthetase, respectively. Bands
2, 9, and 16, which were cut from P1 lanes, were confirmed to contain
glycine tRNA synthetase, while bands 4, 11, and 18, which were cut
from P3 lanes, were confirmed to contain alanine tRNA synthetase,
as expected ( Figure S3 ). The P2 and P4
sera were obtained from patients with scleroderma and were known to
contain anti-topoisomerase I antibodies. − Bands 3, 6, 10, 13, 17, and 20, which were cut from
the P2 and P4 lanes, were all confirmed to contain topoisomerase I
as expected. These data clearly confirmed that the various IP methods
isolated the expected proteins, but importantly the ProMTagged products
appeared to be the most contaminant- and immunoglobulin-free. 

Systemic sclerosis (SSc) encompasses a group of
autoimmune diseases characterized by tissue fibrosis, vascular disease,
and autoantibody production. The most prevalent SSc-related autoantibodies
include anti-centromere, anti-topoisomerase I (ATA), and anti-RNA
polymerase III. Importantly, these autoantibodies mark distinct subsets
with varied clinical phenotypes and disease outcomes/prognoses. As
an example of these phenotype–serotype associations, ILD is
the most common cause of death in SSc patients and is more prevalent
among patients positive for ATA, demonstrating that ATA is an indicator
of SSc prognosis. Accurate autoantibody
identification is therefore crucial for optimal clinical management. Currently, IP is the gold standard method for identifying most
SSc-specific autoantibodies, but the reliability of this assay in
determining the presence of ATA is questionable given that topoisomerase
I appears at the same band size as multiple other human proteins/target
antigens. Ouchterlony DID represents an alternative method of detecting
ATA in SSc patient sera, but there has been little research on its
comparative accuracy and reliability relative to IP or other highly
sensitive, but potentially less specific, commercial methods such
as ELISA. To address these questions, we compared the accuracy
of conventional
IP, ELISA, DID, and IP-to-MS in the detection of ATA in SSc patients.
Sera derived from a group of SSc patients from the University of Pittsburgh
longitudinal cohort previously classified as ATA positive (ATA+) by
conventional IP, DID* (calf thymus extract used as substrate), or
commercial ELISA were reassessed by custom ELISA and DID assays using
recombinant human topoisomerase I as substrate antigen. We assessed
82 serum samples through our custom ELISA. As shown
in Table S2 , 66/82 samples exceeded the
threshold of 0.15 arbitrary units (AU), which was established based
on the mean standardized antibody level (+2 SD) in a healthy control
cohort. Based on the distribution of ELISA values across the full
cohort, we further stratified patients into three groups: high titer
(≥30th percentile rank of cohort), low titer (<30th percentile),
and negative (<0.15 AU). When assessed by our custom Ouchterlony
DID assay, 63/82 samples
yielded precipitin lines consistent with the presence of ATA ( Table S2 ). Five of the 19 samples that were negative
by our custom DID were low titer positive ATA by our ELISA. Of the
66 samples with ELISA evidence of ATA, 61 generated DID patterns indicative
of ATA positivity, and 61/63 samples positive for ATA by DID were
also positive by ELISA ( Table S3 ). The
overall concordance of ELISA and DID was 91%. Based on the assumption
that DID represents the conventional “gold standard”
for accurate detection of ATA, the relative sensitivity and specificity
of our custom ELISA was 0.968 and 0.667, respectively. Because 18%
(15/82) of the cohort previously classified as ATA+ by conventional
IP was classified as ATA negative (ATA−) by DID, our results
suggested that conventional IP alone is unreliable in accurately identifying
ATA positivity. To address this apparent discrepancy, we selected
13 SSc+ patient
sera with discordant ATA assessments (ATA+ by conventional IP, DID*,
or commercial ELISA, but ATA– by our custom DID) for analysis
by IP-to-MS ( , P1–P8, P10, P12, P13, P15, and P16; and Table S2 ). Six sera from SSc+ patients that were deemed ATA–
by conventional IP or commercial ELISA were included as an SSc+ ATA–
control group ( , P9, P11, P14, and P17–P19). In addition, seven ATA+ sera
(positive by conventional IP, as well as our custom ELISA and DID
assays) were selected as reference samples ( , P20–P26). Sera from six healthy
individuals comprised the negative control group ( , P27–P32). With IP-to-MS, quantifiable
topoisomerase I was detected
in 13
samples ( , A) based on spectral
counts of unique topoisomerase I peptides. All of the reference samples
(7/7) were high titer. Six of the ATA– (by custom DID) samples
(6/19) also had detectable topoisomerase I by IP-to-MS; five were
strongly positive and one was weakly positive. Topoisomerase I was
undetectable in 13 of the test samples (13/19), all of which were
also ATA– by both ELISA and DID. Importantly, none of the healthy
control sera contained topoisomerase I reactivity. Overall, there was very good concordance between
the four immunoassays
among the reference samples, with the exception of P20 that demonstrated
low titer positivity by ELISA, but high titer positivity based on
IP-to-MS spectral counts. Among the test group, 5 samples demonstrating
low titer positive ATA by ELISA yielded high-titer positive results
by IP-to-MS. One sample (P6) was negative by ELISA and DID but tested
positive by IP-to-MS. Collectively, these results indicate that IP-to-MS
may be a more sensitive measure of anti-topoisomerase I antibodies
than conventional IP, DID, or ELISA. IP-to-MS provides a survey
of the target cell lysate’s whole
proteome. Figure S4 shows a heatmap of
proteins captured by the IP-to-MS workflow for the 32 sera examined.
As in Figure S2 , there were nonspecific
binding proteins detected in the reference, test, and healthy control
sera. Topoisomerase I was not the only protein specifically precipitated
by the reference and test sera. These other proteins included argonaute
subunits, TRIM21, DDX18, and dihydrolipoyllysine acetyltransferase
( B), demonstrating
that IP-to-MS can also serve as an autoantigen discovery tool. As
further evidence of this diagnostic utility, the test sera that were
topoisomerase I negative contained antibodies to novel antigens, including
coilin, nibrin, MRE11, Nup50, elongator proteins, RNA polymerases,
and exosome subunits ( C). Taken together, these data show that IP-to-MS may
outperform conventional
IP, DID, and ELISA in identifying sera that contain ATA. Equally important,
IP-to-MS has the capacity to identify additional, potentially novel/undiscovered
autoantigens recognized by patient sera, allowing for further stratification
of patients than is possible with conventional methods of autoantibody
detection. 

We present here a novel
IP-to-MS method
for detection of disease-associated
autoantibodies that is founded on coupling ProMTag, a reversible protein
tag that forms a rapid irreversible covalent bond to a bead-based
matrix using click chemistry, to an unbiased pool of potential target
proteins. ProMTagged protein lysate is coincubated with antibodies
from patient sera bound to protein A resin to capture autoantigens.
After washing away unbound proteins, the ProMTagged autoantigens are
released under strong denaturing conditions along with the immunoglobulins
that were bound to the protein A resin. The ProMTagged autoantigens
are separated from the large excess of immunoglobulins by covalent
coupling to click chemistry resin using the pairing of MT and TCO.
Finally, the autoantigens are released from the ProMTag, digested
with MT-Trypsin, and analyzed by MS. We have previously shown
that ProMTag serves as a universal protein
tag which is capable of capturing proteins from a wide variety of
sources. We have also previously shown
that MT-Trypsin is able to rapidly digest proteins while being tethered
to TCO resin, thus enabling the addition of relatively large amounts
of trypsin that do not overly contaminate peptide samples. We show
here that ProMTag is suitable for labeling native proteins and that
the linkage is maintained over hours long incubation times. We also
show that the IP-to-MS method is highly reproducible and faithfully
captures known autoantigens from previously characterized patient
sera. A “real world” test of the IP-to-MS workflow
of 37
samples consisting of 15 positive controls containing autoantibodies
of known specificity, 5 negative controls, and 17 test samples (“unknowns”)
derived from patients with CTD-ILD provided a wealth of information.
All but one positive control yielded the expected autoantigen target,
and all of the test samples yielded candidate autoantigen targets.
Several of the test sera autoantigens matched those recognized by
sera from positive control samples. Because the ProMTag was added
under native conditions, many of the IPs contained protein complexes.
Although our current IP-to-MS method does not distinguish between
direct and indirect autoantibody binding, future development of this
workflow will optimize conditions for mild complex disruption to identify
the direct autoantigen targets. Data from IP-to-MS studies will
be useful for content-rich, molecular
characterization of autoimmune diseases. The current state-of-the-art
approach to autoimmune disease diagnosis includes a combination of
clinical evaluation and targeted autoantibody testing but is limited
to assessment of known autoantigens included in commercially available
tests that are often ELISA-based. Seropositive samples based on ELISA
assays are scored by magnitude of reactivity, while IP, cytometric,
and immunodiffusion assays mostly yield binary positive or negative
results that are difficult to quantify with precision. The identification
of new autoantigens traditionally requires in-depth
bench-side analysis involving multistep, cumbersome methods such as
immunoblotting followed by gel matching, band excision, and MS analysis.
Novel autoantigen discovery has required sophisticated experimental
techniques and research expertise. The IP-to-MS workflow bypasses
many of the complex steps required for novel autoantigen discovery.
The entire workflow takes ∼6 h to complete and is technologically
simple, involving routine microcentrifuge spin-isolation handling.
IP-to-MS also obviates the need for targeted autoantigen tests since
the method is unbiased and can be targeted to any proteome source. A key advantage of the IP-to-MS method is that it produces quantitative
data that can be entered into public data repositories for interstudy
comparisons. This will allow for quantitative longitudinal analysis
to follow the progress of a disease and evaluate the efficacy of a
treatment regime. It also enables large-scale, study-to-study comparisons
for in-depth statistical analysis. The data analysis presented here
only required commonly used analytical tools. Collecting larger data
sets will allow for machine-learning and AI approaches to assess these
quantitative data. Finally, the IP-to-MS workflow is organism,
disease, and condition
agnostic, as it can be applied to any IP experiment and is not limited
by source of antibody or type of cell/tissue extract/substrate. Of
particular interest, this method will have important applications
in other disciplines such as cancer immunology, as all cancers elicit
an immune response. , Harnessing the IP-to-MS method
to identify patient-specific cancer antigens will provide an additional
level of molecular characterization of a tumor, as well as a measure
of cancer load, treatment risk (e.g., immune checkpoint inhibitor-induced
adverse events), treatment response, and relapse, effectively establishing
the foundation for development of personalized treatment regimes. 

