synovial fluid is present in all joint 
 cavities , where it protects 
 the articular cartilage surfaces , in part by reducing friction .
synovial 
 fluid , furthermore , facilitates the transport of nutrients and waste 
 products including proteins and metabolites between the vascularized 
 synovium and the avascular cartilage .
many components of synovial 
 fluid are derived from blood plasma , and these two body fluids share 
 many similarities in terms of protein composition . however , 
 synovial fluid also contains proteins secreted from the surrounding 
 tissue , including the articular cartilage and synovium .
the protein concentration in synovial fluid from 
 healthy knee joints is approximately 25 mg / ml , i.e. , /3 of the concentration found in blood plasma , 
 and albumin constitutes approximately 12 mg / ml .
joint diseases , in particular osteoarthritis 
 ( oa ) and rheumatoid 
 arthritis ( ra ) , are the leading cause of disability in people over 
 55 years .
it has been estimated that , 
 as of 2005 , 27 million adults in the united states have clinical oa , 
 and in 2009 , oa was the fourth most common cause of hospitalization .
furthermore , joint injuries , such as anterior cruciate tear , that 
 predispose to precocious joint failure have become epidemic in young 
 athletes .
although changes in the composition of synovial 
 fluid have been described in patients with joint disease , there do 
 not exist reliable biomarkers for early disease diagnosis or biomarkers 
 that accurately depict response to therapy . as a consequence ,
is in direct contact with the 
 joint tissues , it provides an attractive source of biomarkers candidates 
 for monitoring joint health and for furthering the understanding of 
 the disease mechanisms .
however , 
 the protein concentration , content , and volume of synovial fluid is 
 known to change dramatically during active joint diseases , and few 
 studies have been focused on synovial fluid in healthy state or the 
 likely origins of the synovial fluid proteins .
balakrishnan 
 et al . compared the human synovial fluid 
 proteome from oa and ra patients .
synovial fluid was immune - depleted 
 of the most abundant proteins , followed by liquid chromatography 
tandem 
 mass spectrometry ( lc  ms / ms ) analysis , which led to the identification 
 of 575 proteins , of which 135 demonstrated a greater than 3-fold abundance 
 change between the two groups . in a similar study , mateos et al . identified a total of 136 different proteins 
 using two - dimensional polyacrylamide gel electrophoresis ( 2d - page ) 
 protein separation , followed by in - gel trypsin digestion and protein 
 identification by lc  matrix - assisted laser desorption / ionization 
 time - of - flight ( maldi tof / tof ) ms .
when assessing the total number 
 of different proteins to be expected in synovial fluid , analyzing 
 2d - page images seems to be a valid approach .
analyzed synovial fluid from patients with joint 
 swelling using several precipitation strategies and detected 456 protein 
 spots on 2d - page images .
detected 1000 protein spots on 2d - page images of synovial fluid 
 from ra patients , which was estimated to represent 300 individual 
 proteins .
however , neither of the two latter studies identified the 
 proteins within the protein spots .
2d - page in - gel digestion strategies , 
 while highly informative , are not suitable for high - throughput proteome 
 analyses . therefore , we investigated 
 the use of in - solution proteomics strategies with the aim to increase 
 high - throughput protein identification and quantitation . in this normative 
 proteomics study of synovial fluid from healthy porcine knee joints , 

we report results from applying our optimized method , using porcine 
 synovial fluid . domesticated or minipigs are a highly relevant anatomically 
 large animal model organism to study several human diseases , including 
 acute joint injury , oa , and other inflammatory disease .
the homology between the porcine genome and the human genome is 
 conserved to a much greater extent than that between human and mouse 
 or other rodents .
furthermore , the porcine 
 model has previously been used to study anterior cruciate ligament 
 injury and surgical and tissue engineering approaches for healing .
we also report mrna sequence data from healthy porcine synovium 
 that we used to estimate the contribution to the synovial fluid proteome 
 from this tissue , and we compare this to the relative contribution 
 from plasma ( e.g. , proteins secreted into plasma by the liver or by 
 blood cells ) and articular cartilage .
six 
 adolescent yucatan minipigs ( coyote cci , douglas , ma ) , aged 1215 
 months , were obtained for use in this study .
all minipigs were housed 
 and monitored by the animal resources at boston children s 
 hospital ( arch ) and handled according to approved institutional animal 
 care and use committee ( iacuc ) protocols .
minipigs were acclimated 
 to the arch environment for a minimum of 3 days prior to experimental 
 handling .
nonbloody synovial fluid was obtained from the joints 
 by aspiration using a 21 gauge needle .
samples were centrifuged at 
 3000 g at room temperature for 10 min to pellet and 
 remove cells and cellular debris . in some cases ,
3 ml of sterile saline 
 was injected into the knee joint to facilitate fluid extraction ; after 
 saline injection the knee was bent 10 times to ensure homogeneous 
 fluid distribution and mixing .
furthermore , a human synovial fluid 
 sample was obtained from a ra patient according to an approved irb 
 protocol ( irb - p00006443 ) to evaluate the integrity of the uniprot sus scrofa protein database .
euthanasia of 
 the animals was induced by intramuscular injection 
 of atropine ( 0.04 mg / kg ) , telazol ( 4.4 mg / kg ) , and xylazine ( 2.2 mg / kg ) 
 and finalized by intravenous injection of fatal plus ( 86 mg / kg ) . at 
 the time of euthanasia ,
care was taken to sample only the synovial membrane 
 without any subintimal structures , such as fat or blood vessels .
each 
 tissue specimen was snap frozen in liquid nitrogen and stored at 80 
 c .
total protein concentration for 
 each sample ( diluted 1:30 in water ) was determined for normalization 
 of sample material using a colorimetric ( bradford ) protein assay kit 
 ( bio - rad , hercules , ca ) according to the manufacturer s instructions , 
 with bovine serum albumin used as the standard .
thirty micrograms of total synovial fluid 
 protein was prepared for sodium dodecyl sulfate ( sds)-page in laemmli 
 sample buffer ( bio - rad , hercules , ca ) according to the manufacturer s 
 instructions .
seeblue plus2 pre - stained standard ( invitrogen , carlsbad , 
 ca ) was used as the protein molecular weight standard .
the sample 
 was fractionated using nupage 412% bis - tris minigels ( invitrogen ) 
 at 150 v for 65 min in mops sds - running buffer ( invitrogen ) .
the gel 
 was stained using coomassie blue , simplyblue safestain ( invitrogen ) , 
 according to manufacturer s instructions .
three trypsin digest 
 protocols were evaluated : performed using the fasp protein digestion 
 kit ( protein discovery , 
 san diego , ca ) according to manufacturer s instructions using 
 30 kda cutoff spin filters .
ninety micrograms of total synovial fluid 
 protein was digested overnight at 37 c with 2 g of sequencing 
 grade modified trypsin ( promega , fitchburg , ma ) . to assess the need 
 of glycan removal
when working with synovial fluid , 500 u peptide - n4-(n - acetyl - beta - glucosaminyl)-asparagine 
 amidase ( pngase f ) ( new england biolabs , ipswich , ma ) was added to 
 these samples prior to the trypsin digestion step , and samples were 
 incubated overnight at 37 c , after which the normal fasp protocol 
 was continued .
after trypsin digestion , the samples were desalted 
 with targa c18 columns ( nest group , southborough , ma ) and resuspended 
 in 5% acetonitrile ( acn ) and 5% formic acid ( fa ) prior to analysis . performed 
 according to gallien et al .
ninety micrograms 
 of total synovial fluid protein was diluted with 8 m urea in 100 mm 
 ammonium bicarbonate to a final volume of 25 l .
the sample 
 was reduced with dithiothreitol at a final concentration of 12 mm 
 for 30 min at 37 c and alkylated with iodoacetamide at a final 
 concentration of 40 mm for 1 h at room temperature in the dark .
the 
 samples were diluted with 100 mm ammonium bicarbonate to a total volume 
 of 100 l , 2 g of trypsin was added , and the sample was 
 digested overnight at 37 c .
the samples were desalted with targa 
 c18 columns ( nest group ) and resuspended in 5% acn , 5% fa prior to 
 analysis .
three gel - lanes , 
 each loaded with 150 g total synovial fluid protein , were divided 
 into 10 sections each and subjected to standard in - gel tryptic digestion 
 as previously described , followed by analysis .
human plasma was acquired 
 as part of an ongoing method optimization study using a deidentified , 
 discarded cord plasma sample and thus are not considered research 
 of human subjects .
one hundred micrograms of plasma protein was digested 
 using the fasp protein digestion kit ( protein discovery , san diego , 
 ca ) with a modification to the recommended protocol .
this modification 
 involved the use of a 10 kda mwco filter instead of the stock 30 mwco 
 filters .
a trypsin / lysc mix ( promega , madison , wi ) was added to the 
 fasp filter at 1:25 ratio for digestion .
the samples were then incubated 
 at 37 c overnight , and the resulting peptides were recovered 
 as recommended by the manufacturer s protocol .
the peptides 
 were desalted using oasis hlb columns ( waters , milford , ma ) and resuspended 
 in 2% acn in 0.1% fa prior to analysis .
two different high - resolution / high - accuracy 
 mass spectrometer systems 
 were used for the shotgun proteomic analysis : ( 1 ) for post - translational 
 modification ( ptm ) analysis and method optimization , synovial fluid 
 samples were analyzed on a tripletof 5600 ( ab sciex , framingham , ma ) 
 connected online with a nanoflow uplc and a nanoflex system ( eksigent / ab 
 sciex ) .
the samples were loaded onto a 15 cm reversed - phase c18 200 
 m chip with 2 l / min in 100% solvent a ( 0.1% fa ) .
the 
 samples were then separated using a 15 cm reversed - phase c18 75 m 
 chip and eluted with a linear gradient of 2% solvent b ( 0.1% fa in 
 acn ) , which was raised to 35% solvent b over 120 min ( 60 min for in - gel 
 digested samples ) at a constant flow rate of 500 nl / min .
( 2 ) the six 
 fasp digested synovial fluid samples used to determine the synovial 
 fluid protein list and abundances as well as the trypsin - digested 
 human plasma samples were analyzed on a q exactive ( thermo scientific , 
 waltham , ma ) connected online to an easy - nuplc 1000 ( thermo scientific ) . 

the samples were loaded onto a 10.5 cm reversed - phase c18 picochip 
 with a flow rate of approximately 1 l / min in 98% solvent a 
 and 2% solvent b and were eluted with eluent b using a linear gradient 
 that was raised to 35% over 120 min at a constant flow rate of 300 
 nl / min .
1656 , paragon algorithm 4.5.0.0 ) . to identify the most commonly 
 single observed ptms ,
data files were searched in thorough mode with 
 a focus on biological modifications in proteinpilot to include more 
 than 300 different ptms .
the .raw data files from synovial fluid 
 and human plasma analyzed 
 on the q exactive were searched using maxquant 1.4.1.2 .
all standard settings were employed with carbamidomethyl 
 ( c ) as a static modification and deamidation ( nqr ) , oxidation ( m ) , 
 and protein n - terminal acetylation included as variable modifications . 

label - free quantitation of all proteins was performed in maxquant 
 based on integrated precursor intensities .
protein abundances are 
 represented as protein intensity - based absolute quantitation values 
 ( ibaq ) and are reported for all proteins having at least two quantifiable 
 unique peptides in at least three lc  ms runs .
the human plasma sample was searched against the 
 uniprot homo sapiens reference proteome 
 database with isoforms 
 ( downloaded 7/18/2014 , containing 89  032 entries ) .
the porcine 
 synovial fluid data was searched against the uniprot s. scrofa reference proteome database ( downloaded 
 11/09/2013 , containing 26  070 entries ) .
the human ra synovial 
 fluid , used to evaluate the uniprot s. scrofa database , was searched against all reviewed h. sapiens uniprot proteins ( downloaded 08/10/2013 , containing 20  277 
 entries ) .
all proteins and peptides are reported below a 1% false 
 discovery rate ( fdr ) cutoff , and protein posterior error probability 
 ( pep , equivalent to expectancy ) was investigated to ensure only confident 
 protein identifications .
for the ptm 
 analysis , the search results were analyzed using proteinpilot descriptive 
 statistics template , version 3.001 , and for the protein abundance 
 analysis , the ibaq values were analyzed using perseus , version 1.4.1.3 , 
 and ibm spss statistics ( version 21 ) .
four criteria were required to assign n - glycosylation 
 sites : ( i ) 
 a 1% fdr cutoff to all peptide spectral matches ( psms ) ; ( ii ) all site 
 assignments required the presence of a consensus site ( cs ) for n - glycosylation , 
 i.e. , nx(s / t ) , where x may be any amino acid except proline ; ( iii ) 
 once cs status was established for all peptide assignments , an asparagine 
 deamidation at the asparagine within the cs was required ; and ( iv ) , 
 finally , all true site assignments were required to come from sample 
 preparations that were treated with pngase f. the fdr of site assignment 
 was estimated by evaluation of the random rate of site assignment 
 among control samples that were not treated with pngase f. in this 
 way , the rate of psms leading to the identification of a deglycosylated 
 peptide may be compared .
total rna was extracted 
 from frozen 
 synovium tissue using the purelink rna mini kit ( ambion , austin , tx ) , 
 treated with purelink dnase i ( life technologies , grand island , ny ) 
 according to the manufacturer s protocol , and quantified .
briefly , 
 frozen tissue samples were placed in tubes containing 5 metal lysing 
 beads ( metal bead lysing matrix , mp biomedicals , solon , oh ) and 1 
 ml of trizol ( life technologies ) .
tissue was homogenized using a fastprep24 
 ( mp biomedicals , solon , oh ) at 6 m / s for 40 s. this was repeated two 
 additional times , with samples briefly placed in liquid nitrogen between 
 homogenization runs .
following total rna extraction and quantitation 
 via spectrophotometry using a nanodrop 1000 ( thermo scientific , wilmington , 
 de ) , samples were submitted for quality analysis on a bioanalyzer 
 model 2100 ( agilent technologies , santa clara , ca ) .
each rna sample 
 had an rin > 7.20 , indicating that they were of sufficient quality 
 for sequencing .
a mrna library for each tissue 
 sample was prepared as previously described using the truseq rna sample 
 preparation kit , v2 ( illumina , san diego , ca ) .
briefly , mrna was enriched from total rna , chemically fragmented , 
 reverse - transcribed using random hexamers , and ligated to barcoded 
 adapters per the manufacturer s instructions .
the cdna fragments 
 were amplified via pcr , and the libraries were washed with ampure 
 xp beads ( beckman coulter inc . , danvers , ma ) to remove primer dimers . 

one microliter aliquots from each library were run on a 420% 
 tbe gel ( life technologies , grand island , ny ) for verification .
equal 
 amounts of dna from separately barcoded cdna libraries were pooled 
 ( n = 12 per lane ) and sent for 50 base pair paired - end 
 sequencing on an hiseq 2000 ( illumina ) .
known genes 
 were annotated with an r script based on data available from ensembl . 

expression level of each gene was quantified by the reads per kilobase 
 of exon per million mapped reads ( rpkm ) .
the repeatability of the 
 rna - seq data was measured by comparing libraries generated from the 
 right and left knees of the same animal and calculating the pearson 
 correlation coefficient ( r ) with respect 
 to the genome - wide rpkm values .
the calculated rpkm values were averaged 
 across 12 samples for each gene , and these mean values were used in 
 ranking the genes with respect to their expression levels . in 
 order to estimate the likely source of proteins detected in the synovial 
 fluid , we additionally reviewed mouse liver , mouse blood , and human articular cartilage 
 ( authors unpublished data ) rna - seq data .
on the basis of homology 
 information retrieved from the ensembl database , we matched average 
 rpkm values for each transcript with the proteins detected in the 
 synovial fluid , and we identified the proteins for which all four 
 mrna expression values were available .
we then determined the subset 
 of proteins with a signal peptide . among these proteins , we identified 
 those with at least 2-fold higher mrna levels in the synovium compared 
 to all of the other individual tissues , and we then performed the 
 same calculations for liver , cartilage , and blood .
initially , 
 we determined whether precipitating the synovial fluid 
 proteins increased the diversity of the detectable protein bands by 
 coomassie - stained sds - page
. however , similar to chen et al . , we found that precipitation appeared to reduce 
 polypeptide diversity rather than increase it ( data not shown ) ; thus , 
 we chose not to use precipitated samples for our subsequent analyses . 

likewise , because albumin can function as a protein carrier for a 
 wide range of proteins , we chose not to deplete albumin or other abundant 
 proteins from our samples to avoid changing protein abundance profiles 
 and removing important proteins that might bind the depletion targets .
zhou et al . reported that removing albumin led to the depletion 
 of many proteins in human serum , including clinically useful biomarkers .
we compared urea in - solution digestion to fasp to find the most 
 efficient digestion method without prior prefractionation for synovial 
 fluid and used the robust and proven in - gel digestion as a reference . 

in contrast , fasp and in - gel - based digestion each yielded 
 227 protein identifications , indicating similar performance of the 
 two digestion techniques .
however , 170 of the 227 identified proteins 
 were found in at least two of the three synovial fluid samples when 
 using the fasp protocol in contrast to only 145 of the 227 proteins 
 in the case of the in - gel digestion protocol ( figure 1 ) .
furthermore , despite substantial overlap among the data 
 sets , roughly 26% of proteins found in at least two of the three synovial 
 fluid samples with fasp were missed by in - gel digestion , and 12% of 
 the in - gel digestion proteins were missed with fasp . additionally , 
 17% of the proteins unique to the in - gel digestion were keratins , 
 supporting the notion that in - gel digestion protocols can be prone 
 to keratin contamination .
besides this difference , no other bias was 
 found with regard to which groups of proteins were identified uniquely 
 with the different digestion methods .
this indicates that the fasp 
 protocol gives more consistent data than the in - gel digestion protocol . 

it is possible that depletion strategies can increase the number of 
 identified proteins further ; however , this was not investigated in 
 this study .
number of proteins uniquely identified 
 in at least two of the three synovial fluid samples , using fasp digestion , 
 urea in - solution digestion , and in - gel digestion .
the uniprot s. scrofa reference 
 proteome is not as well annotated as the human proteome .
therefore , 
 in order to determine whether our proteomic method is as sensitive 
 for detecting pig synovial fluid proteins as it would be for human 
 synovial fluid proteins , we performed the same fasp digestion protocol 
 on a synovial fluid sample from a ra patient . in
the four technical 
 replicates , we identified , on average , 173 different proteins in the 
 human sample , which seems to be comparable to the 179 different proteins 
 found , on average , in the individual porcine synovial fluid fasp lc 
the protein composition of synovial fluid is known to be altered 
 during active joint diseases , so the protein overlap was not investigated .
nonetheless , the similar number of identified proteins with the 
 two databases indicates that the uniprot s. scrofa reference proteome is adequate for the analysis of the porcine synovial 
 fluid .
many ptms occur in vivo or are 
 introduced during sample preparation prior to ms analysis in vitro .
therefore , to identify which ptms should be considered for the database 
 search of ms data from fasp - digested synovial fluid samples , we analyzed 
 the identified peptides from this method in proteinpilot descriptive 
 statistics template ( table 1 ) .
several ptms 
 were identified , and all were likely artifacts from the sample preparation . 

chloroacetamide can be used as an alternative alkylating agent to 
 iodoacetamide to reduce the number of alkylation related artifacts . however , due to the low number of detected peptides 
 with these artificial modifications , this was not investigated further .
synovial fluid is known 
 to be rich in glycoproteins , which prompted 
 us to evaluate the effect of removing n - linked glycans on the number 
 of identifiable peptides and proteins .
we compared two synovial fluid samples using the fasp protocol , 
 with and without deglycosylation by pngase f (  ) .
as expected , 
 psms resulting in identification of deglycosylated peptides considered 
 to be previously n - glycosylated was increased 40-fold in the 
 pngase f - treated samples in comparison to that of the untreated samples 
 ( table 2 ) .
twenty - eight and 34 unique deglycosylated 
 peptides in the biological repeats were identified using the four 
 criteria described in the experimental procedures section , compared to only two peptides in each of the untreated 
 samples ( supporting information table 1 ) .
however , all deglycosylated peptides belonged to proteins that 
 had already been identified from other peptides and thus resulted 
 in no new protein identifications .
twenty - eight and 34 unique deglycosylated 
 peptides are relatively low numbers for an analysis of a biological 
 fluid known to be rich in glycoproteins , which may be due to a number 
 of factors : first , the proteome of synovial fluid may not be particularly 
 complex .
this hypothesis is consistent with the proteomics data we 
 observed in this study and with previously reported 2d - page analyses .
n - glycosylation , which 
 is the only glycosylation amenable to pngase f treatment protocols , 
 may not be highly prevalent in synovial fluid .
synovial fluid contains 
 significant amounts of glycosaminoglycans , possibly lessening the 
 biologic necessity for n - glycosylation .
third , the 
 finite dynamic range of the ms analysis might cause abundant peptides 
 to hinder the detection of less abundant ones .
a study that exclusively 
 focuses on o - glycosylation in synovial fluid and combines data from 
 depleted and nondepleted samples will likely identify more extensive 
 glycosylation than that reported here . in order to identify 
 synovial fluid proteins that are secreted by 
 the synovium cells ,
i.e. , transcribed and translated , we generated 
 sequencing data of mrna as active transcripts for pig synovium .
we 
 prepared and sequenced knee synovium from the right and left legs 
 of 6 pigs . on average
, we obtained 12.4 million sequencing reads per 
 library , of which 90% mapped to the pig genome and 81% aligned uniquely . 

we then calculated rpkm values for individual genes and compared their 
 expression level within each individual animal s left and right 
 knee ( figure 2a ) .
intra - animal pearson correlation 
 coefficients exceeded 0.85 , indicative of reproducible data ( figure 2d ) .
the top 20 abundant protein - coding mrnas that 
 did not originate from the ribosome or mitochondria exhibited minimal 
 variation in abundance , as they ranked in the top 1% of all sample - specific 
 rna - seq data sets that we analyzed ( figure 2c ) .
rna - seq of pig synovium is repeatable and indicates high expression 
 of several transcripts encoding secreted proteins .
( a ) table indicating 
 the total number of reads and percentages of successfully mapped reads . 

more than 90% of reads on average are mapped to the pig genome ( susscr3 ) , 
 and more than 80% of reads are mapped uniquely .
( b ) top 20 protein 
 coding genes ( based on expression level measured with rpkm ) that do 
 not originate from the mitochondria or the ribosome .
( c ) box plot 
 indicating the variation in the expression - based rank order of genes 
 listed in ( b ) in all 12 libraries analyzed .
the highest variation 
 is in the rank of prg4 ; however , it remains within the top 1% of genes 
 in all libraries .
( d ) scatterplots indicating high similarity ( r > 0.85 ) between the synovial transcriptomes 
 of paired left and right legs of all pigs .
transcripts for the secreted proteins 
 decorin ( dcn ) and lubricin ( prg4 ) , red blood cell - derived beta hemoglobin 
 ( hbb ) , an adipocyte - derived transcription factor ( pparg ) , and secreted 
 protein leptin ( lept ) are indicated with red colored symbols and closely 
 follow the y = x line ( solid red 
 line ) in each plot .
importantly , transcripts for matrix - degrading 
 enzymes and inflammatory cytokines are not abundant , clustering in 
 the bottom left corner of the panels .
focusing on transcripts encoding proteins containing signal 
 peptides , 
 which are most likely secreted or targeted to cell surfaces , we observed 
 high expression of known connective tissue proteins such as decorin 
 ( dcn ) , fibronectin ( fn1 ) , collagen type iii ( col3a1 ) , and clusterin 
 ( clu ) ( figure 2b ) .
importantly , the principal 
 lubricating protein in synovial fluid , lubricin ( prg4 ) made by type 
 b synoviocytes , was also highly expressed . from these data
, we produced 
 a pig synovium transcriptome database ( supporting 
 information table 2 ) , which is composed of transcripts encoding 
 known and predicted proteins .
we opted for the fasp digestion 
 strategy for the detailed triplicate 
 proteomic analysis of synovial fluid from 6 healthy pigs .
this choice 
 was based on the number of identified proteins , sample amount requirement , 
 processing time , and instrument time .
we identified 374 different 
 proteins in total using the uniprot s. scrofa database and the synovium transcriptome database , and 42 proteins 
 were identified solely in the transcriptome database .
this seemed 
 to be a large fraction assuming an adequate performance of the uniprot 
 pig database , so all identified proteins were sorted by pep ( equivalent 
 to expectancy ) , with a high pep value 
 indicating a lower confidence in the identification ( figure 3a ) .
proteins unique to the transcriptome database 
 displayed high pep values , indicating that the number of identified 
 proteins is too low for the global fdr calculation . to address this 
 issue , all proteins with pep > 1  10 were 
 removed from further analysis , which came to 92 identified proteins , 
 of which 36 were unique to the transcriptome database .
furthermore , 
 we manually assessed the fragment spectra for proteins identified 
 based on a single peptide and removed poor spectra with many unassigned 
 high - intensity signals , which resulted in one additional protein being 
 removed .
after these two filtering steps , 267 proteins remained , of 
 which 6 were unique to the transcriptome database ( figure 3b ) .
five of the 6 unique proteins were immunoglobulin 
 lambda like proteins ( upid : iglv-7 to iglv-11 ) , and the identified peptides 
 originate from a variable region of the lambda chain , which is not 
 annotated in the uniprot database .
the remaining protein , ribonuclease 
 4 ( upid : i3ldz2_pig ) , was present in the uniprot database , but it had not been identified . 

we included several keratins ( 10 , 14 , 3 , and 75 ) in the synovial fluid 
 proteome because keratin - coding mrna was detected in the articular 
 cartilage .
however , we can not preclude some of the keratins being 
 contaminants from sample preparation . analysis of identified synovial fluid 
 proteins . ( a ) identified 
 proteins at 1% fdr sorted by pep .
proteins unique to the transcriptome 
 database ( red squares ) were , for the most part , identified with low 
 confidence ; hence , all proteins with pep > 1  10 ( red line ) were removed from further analysis .
( b ) synovial fluid 
 proteins identified using the uniprot database and the transcriptome 
 database fulfilling the filtering criteria . of the 267 synovial fluid 
 proteins , 140 had rpkm > 5 in the synovial mrna libraries , indicating 
 they are expressed by synovium . of the identified 267 different proteins in synovial fluid 
 ( supporting information table 2 ) , 194 proteins 
 ( 73% ) were identified in all pigs , indicating similar protein expression 
 patterns between the pigs and a high method robustness .
the difference 
 in identified proteins between the pigs likely originates from biological 
 diversity , which is to be expected , and the finite dynamic range of 
 the ms analysis . to investigate the relative 
 protein abundances in synovial fluid , we investigated individual synovial 
 fluid protein abundance by calculating ibaq values .
the protein abundance in synovial fluid , as estimated by 
 the 203 ibaq quantifiable proteins , spans 5 orders of magnitude ( figure 4 ) . as expected , albumin was the most abundant protein , 
 and no apparent bias could be found regarding the abundances of serum - derived 
 proteins ( rpkm < 5 ) compared to proteins expressed by the synovium 
 in the joint ( rpkm > 5 ) ( see below ) .
the relative proportion of proteins whose mrna transcript abundances 
 were less than or greater than 5 rpkm in the synovial membrane transcriptome 
 are colored blue and yellow , respectively .
synovial fluid is 
 a transudate of blood that has additional components added by the 
 surrounding joint tissue including synovium , cartilage , and , in the 
 knee , ligaments and menisci . to determine
whether a likely tissue 
 source for a synovial fluid protein can be predicted , we compared 
 protein expression with the pig synovium transcriptome , a human articular 
 cartilage transcriptome we previously generated , and published mouse 
 liver and blood transcriptomes ( supporting information table 
 2 ) . as expected , albumin in synovial fluid solely derives from 
 blood , as its transcript is highly expressed only in liver ( figure 5 ) .
( a ) tissue - specific histograms indicating the number of detected 
 proteins corresponding to each transcript .
genes are ranked based 
 on mrna expression along the x axis , and the secondary y axis ( in red ) indicates the expression levels in rpkm 
 ( in log scale ) .
( b ) table indicating the top 20 mrnas in synovium 
 with detectable protein products in synovial fluid .
gene expression 
 data derived from published and unpublished studies on tissues with 
 potential influence on protein expression repertoire of serum and 
 synovial fluid are also presented in the three columns on the right . 

blood and liver mrna data originate from mice , and articular cartilage 
 mrna data originate from humans .
( c ) table indicating the top 20 proteins 
 with no detectable mrna expression in the synovium : note that the 
 majority of mrna species corresponding to these proteins are abundantly 
 expressed by liver cells .
when we compared the proteins detected in the synovial fluid 
 with the mrna expression 
 levels in four possible sources , we found that the majority of proteins 
 corresponded to highly expressed transcripts regardless of the source 
 and that liver and synovium likely made the greatest contribution 
 ( figure 5a ) .
further analysis of our data revealed 
 that 171 proteins detected in the synovial fluid had a signal peptide 
 and that liver , blood , and articular cartilage mrna expression data 
 were available for 149 proteins ( supporting information 
 table 2 ) .
of these 149 common proteins in the synovial fluid 
 proteome , 113 most likely derive from plasma transudate since their 
 transcript abundance is 2-fold or higher in liver or blood than that 
 in synovium or cartilage .
in contrast , 37 synovial fluid proteins 
 have transcripts whose expression in synovium is at least 2-fold higher 
 than that in other tissues , while 11 proteins have at least 2-fold 
 or higher transcript expression in articular cartilage .
for example , 
 aggrecan , type ii collagen , cartilage oligomeric matrix protein , and 
 cartilage intermediate layer protein 2 are detected in the synovial 
 fluid , and the corresponding transcripts are expressed at greater 
 levels in articular cartilage than in other tissues .
similarly , proteins 
 involved in post - translational modification of type i collagen ( such 
 as procollagen c - endopeptidase enhancer ) and connective tissue markers 
 ( such as fibronectin 1 and clusterin ) were expressed at higher levels 
 in synovium than in other tissues .
thus , the putative origins of a 
 substantial number of synovial proteome constituents can be predicted 
 from the rna sequencing data .
for those 17 proteins whose mrnas are 
 expressed by multiple tissues , tissue - specific changes in mrna abundance 
 will need to be correlated with changes in synovial protein abundance 
 to reliably determine their most likely tissue of origin .
we 
 have additionally detected several proteins without a signal 
 peptide , which are likely residual fragments that remained in the 
 synovial fluid following apoptosis .
some examples of these are hemoglobin 
 ( hba and hbb ) , which are expressed at extremely higher levels in blood 
 than the remaining tissues , and beta - actin , a ubiquitously expressed 
 protein whose mrna was detected at high levels ( rpkm > 180 ) in 
 all 
 four tissues . also of interest
are proteins not found in healthy pig 
 synovial fluid and expressed at low levels in healthy synovium . increased 
 amounts of matrix metalloproteinases , including mmp13 and adamts4 , 

have been reported in synovial fluid from humans with oa , while proteins 
 involved in the inflammatory / immune response , including complement 
 activation , were found in patients with ra .
these proteins were neither detected in healthy pig synovial fluid 
 nor were their transcripts abundant in healthy synovium .
therefore , 
 in addition to monitoring changes in protein abundance for the healthy 
 synovial fluid proteome , it will be of interest to compare proteomes 
 and transcriptomes of healthy and diseased joints to identify pathways 
 that may be integral to disease processes or mechanisms .
previous 
 comparisons of synovial fluid obtained from patients with ra and oa 
 identified 135 proteins at least 3-fold differentially abundant between 
 the two groups . finally , to 
 identify similarities between synovial fluid and blood plasma , from 
 which
many of the synovial fluid components were found to originate , 
 we characterized the human plasma proteome ( supporting 
 information table 3 ) and identified and quantified a total 
 of 168 human plasma proteins .
the detected ibaq values of the human 
 plasma proteins span 5 orders of magnitude , similar to what was found 
 for the synovial fluid , demonstrating the large degree of protein 
 abundance variation encountered in the two body fluids
. however , the 
 actual range of protein abundances in plasma and synovial fluid is 
 expected to be several orders of magnitude larger , due to the limited 
 dynamic range of the ms analysis . probing 
 the top 10 most abundant proteins in both fluids , the most abundant 
 protein is serum albumin , as expected ( table 3 ) .
other shared high - abundance proteins include serotransferrin , 
 apolipoprotein a , and several immunoglobulins .
serotransferrin and 
 apoliopoprotein a are binding and transporting proteins of iron and 
 lipids , respectively .
the high abundance of the transporter 
 proteins and immunoglobulins points to the shared functions of the 
 two body fluids as a transport media of cellular products .
the main 
 difference between the high - abundance proteins in the plasma and synovial 
 fluid proteomes is the high abundance of hemoglobin in the synovial 
 fluid , which is detected with a lower abundance in plasma ( rank 25 ) . 
 in both fluids ,
the protein is a likely contaminant from red blood 
 cells prior to fluid centrifugation .
fibrinogens , involved in blood 
 clotting , were identified with high abundance in plasma in contrast 
 to that in synovial fluid , as expected . as determined 
 by ibaq values .
our findings 
 demonstrate the high degree of similarity shared by 
 the synovial fluid proteome and the plasma proteome as well as the 
 serum - derived nature of many synovial fluid components .
the expected 
 high degree of similarity found between human plasma and porcine synovial 
 fluid , furthermore , indicates that porcine model systems of synovial 
 joints are suitable for studies focusing on human joint diseases .
we utilized porcine synovial fluid because this animal 
 is used to model human joint disease . in this study , we developed 
 a fasp - based analysis pipeline that consumes only 0.1% of the starting 
 sample material and 10% of the ms instrument time used by , e.g. , balakrishnan ; thus , our method lends itself to clinical proteomics 
 studies on larger cohorts . applying this fast and efficient workflow , 
 we completed a normative proteomics study of synovial fluid from healthy 
 knee joints and identified more than 250 proteins using very stringent 
 identification criteria .
preprocessing with pngase f to remove n - glycosylation 
 was not found to be essential for the proteomic analysis of synovial 
 fluid . through an analysis of high - occurrence peptide ptms
, we found 
 that proteomics projects using the fasp protocol for synovial fluid 
 analysis , and possibly for other biological samples as well , should 
 include peptide n - terminal acetylation , deamidation of asparginine 
 and glutamine , and peptide n - terminal pyro - glutamic acid in the data 
 analysis for comprehensive peptide identification .
ms - based proteome mapping , 
 we also classified and predicted the likely tissue origins for the 
 majority of the detected proteins in healthy synovial fluid by cross - referencing 
 the proteome with the rna transcriptomes from synovium , cartilage , 
 blood , and liver .
although many proteins derive from plasma transudate , 
 as expected , an important subfraction appears to be solely expressed 
 by synovium or cartilage .
changes in the abundance of these latter 
 proteins in synovial fluid or in blood may be useful biomarkers of 
 disease onset or progression .
for synovial fluid proteins whose transcripts 
 are expressed in multiple tissues , it will be necessary to correlate 
 tissue - specific changes in mrna expression with changing synovial 
 fluid protein abundance in order to determine these proteins 
 principal tissues of origin .
finally , as many proteins were found 
 to likely originate from plasma , we compared the identified porcine 
 synovial fluid proteome to the proteome of human plasma .
we found 
 that the two body fluids share many similarities in terms of protein 
 functions and localizations , underlining the detected plasma derived 
 nature of many synovial fluid components .
our methods and the 
 resulting proteome and transcriptome data sets 
 will be useful when comparing porcine synovial fluid in healthy and 
 diseased states .
importantly , the high homology between pigs and humans 
 should make these methods and data sets valuable for human studies .
 synovial 
 fluid in an articulating joint contains proteins derived from the 
 blood plasma 
 and proteins that are produced by cells within the joint tissues , 
 such as synovium , cartilage , ligament , and meniscus . 
 the proteome 
 composition of healthy synovial fluid and the cellular origins of 
 many synovial fluid components are not fully understood . here , 
 we 
 present a normative proteomics study using porcine synovial fluid . 
 using our optimized method 
 , we identified 267 proteins with high confidence 
 in healthy synovial fluid . 
 we also evaluated mrna expression data 
 from tissues that can contribute to the synovial fluid proteome , including 
 synovium , cartilage , blood , and liver , to better estimate the relative 
 contributions from these sources to specific synovial fluid components . 
 
 we identified 113 proteins in healthy synovial fluid that appear to 
 be primarily derived from plasma transudates , 37 proteins primarily 
 derived from synovium , and 11 proteins primarily derived from cartilage . 
 
 finally , we compared the identified synovial fluid proteome to the 
 proteome of human plasma , and we found that the two body fluids share 
 many similarities , underlining the detected plasma derived nature 
 of many synovial fluid components . knowing the synovial fluid proteome 
 of a healthy joint will help to identify mechanisms that cause joint 
 disease and pathways involved in disease progression . 
Introduction
Experimental Procedures
Results and Discussion
Conclusions