in the post - genomic 
 era , next - generation sequencing technology 
 is now widely used to characterize the alterations of genome and transcriptome 
 in the context of human diseases . although 
 gene expression can be analyzed by transcriptomic profiling , transcriptomic 
 data
do not always correlate well with protein expression in biological 
 samples and often lack the information on protein posttranslational 
 modifications .
thus , the development of proteomics platforms for deep 
 proteome coverage becomes an urgent task to provide systematic and 
 comparable protein expression information complementary to dna and 
 rna data .
mass spectrometry ( ms)-based shotgun proteomics is 
 predominantly 
 used for complex proteome analysis . in a typical 
 shotgun experiment ,
complex protein samples extracted from cells or 
 tissues are digested with protease(s ) , and the resulting peptide mixtures 
 are fractionated by organic gradient on hplc columns , followed by 
 tandem mass spectrometry analysis .
the ms / ms spectra are then searched 
 against protein database for the identification of proteins and posttranslational 
 modifications . with the advent of high - resolution ms and the improvement 
 of liquid chromatography ( lc ) performance , the current platform of 
 shotgun proteomics
can routinely identify thousands of proteins in 
 mammalian cells in a single lc  ms / ms analysis .
one of the key 
 measurements of lc performance is the peak capacity that is defined 
 as the number of peaks separated within a resolution of unity in a 
 given lc gradient time .
peak capacity 
 is estimated to be proportional to the root square of lc column length 
 and inversely proportional to the root square of lc particle size .
several reports demonstrated the benefits of 
 small particles ( < 2 m ) with ultrahigh pressure solvent delivery 
 ( up to 70  000 psi ) .
high values of peak capacities were obtained on these 
 columns ( i.e. , 75 m  50 cm ) depending on the gradient 
 length . however , column heating and ultrahigh system pressure ( > 10  000 
 psi ) are usually required for running long columns packed with sub-2 
 m beads , compromising the robustness of the system . alternatively , 

when hplc time is not a limiting factor , longer lc columns improve 
 resolving power but with a higher backpressure .
for instance , several 
 reports have shown comparable peak capacity using 5 m c18 particles and up to 1 m long column within regular hplc 
 pressure limit . when long lc was coupled to q 
 exactive ms , it resulted in more than 4000 identified proteins in 
 the human proteome under optimized conditions . however , because of the large ( > 10 ) dynamic range 
 of 
 proteins in mammalian cells , additional prefractionation step(s ) ( e.g. , 
 sds - page , strong anion exchange ( sax ) , strong cation exchange ( scx ) , 
 basic ph lc and isofocusing ) were applied to reduce peptide complexity 
 and deepen the mammalian proteomic analysis .
further peptide separation was also achieved through 
 gas - phase fractionation ( gpf ) through ms1 ion selection on mass spectrometer . to date , a few studies lead to the detection of more than 
 10  000 
 proteins in several human cancer cell lines using sax fractionation 
 and analysis on ltq orbitrap velos ms and about a month of instrument 
 time .
the marto group identified 11  352 
 mouse genes - derived proteins using ltq xl ms and triple tof 5600 ms 
 in 8 days from murine embryonic stem cells .
lehtio group reported the identification of 13  078 human 
 proteins and 10  637 mouse proteins from cancer cell lines using 
 high - resolution isofocusing fractionation and ltq orbitrap velos ms 
 with 15 days of instrument time .
while we were preparing this manuscript , mann s group reported 
 the identification of 10  000 proteins on q exactive 
 ms using 4 day instrument time and long column coupled to uplc system .
most recently , drafts of the entire human proteome 
 ( identifications of 18  000 genes products in varieties 
 of human tissues and hematopoietic cells ) were completed from 2000 
 lc 
ms / ms runs using several months of ms instrument time by 
 two research groups .
although significant progress 
 has been achieved to identify deep 
 mammalian proteome , there is no systematic report on the adjustment 
 of parameters for ultralong lc 
ms / ms runs to optimize protein 
 identification on a genome wide scale . to obtain an in - depth coverage 
 of mammalian proteome
ms / ms platform , following our previous optimization 
 work using a regular short column ( 75 m  12 cm ) . in this study
, we described a stepwise analysis 
 to tune shotgun proteomics parameters using an in - house manufactured 
 150 cm lc column coupled to q exactive ms .
ms / ms runs of analyzing mammalian 
 tissue ( e.g. , rat brain ) .
finally , we used the optimized lc / lc  ms / ms 
 platform to process a human brain specimen of alzheimer s disease 
 ( ad ) and identified more than 10  000 proteins , covering more 
 than 60% of the expressed proteome .
the capillary 
 column of 150 cm in length and 100 m inner 
 diameter ( i d ) was packed in - house following the previously reported 
 protocol with modifications .
this column 
 consisted of two segments , namely , one 110 cm long blunt end - capillary 
 column and one 40 cm long capillary column with a 15 m opening 
 tip . to make the blunt end column
, we dipped 100 m i d fused 
 silica tubing into the activated silicate solution ( next advance , 
 ny ) , briefly followed by heating to 100 c on a heater plate 
 for 1 min before the ejection of excess silicate solution .
then , the 
 frit was further heated for another hour at 100 c and cut to 
 2 mm in length .
the blunt end column was then packed with slurry of magic c18 aq 200 
 beads ( 5 m ) at a concentration of 30 mg / ml in methanol .
a bed 
 length of 110 cm was obtained after 6 h of continuous packing at 2800 
 psi using a pressure injection cell system ( next advance , ny ) .
the 
 second segment of capillary column was packed similarly to 40 cm in 
 length using self - pack picofrit column ( new objective , 15 m 
 tip opening , 100 m i d , cat no .
finally , two 
 columns were connected through a metal union with zero dead volume 
 ( upchurch scientific , ny ) . human tissues of prefrontal cortical 
 regions were provided by the 
 brain and body donation program at banner sun health research institute . 
 the ad case with short post - mortem interval ( <3 h ) was clinically 
 and pathologically characterized in accordance with established criteria .
adult rat brains were purchased from pel freez 
 biologicals , and rat brain peptides were prepared as previously described .
the cerebral cortex of ad brain was homogenized 
 in 100 l of lysis buffer ( 0.1 m tris , ph 8.5 , 8 m urea , 0.15% 
 sodium deoxycholate ) at 4 c using 0.5 mm glass beads for 5 min 
 in a bullet blender instrument ( next advance ) .
the entire cell lysate without clarification of the insoluble materials 
 was digested with lys - c ( wako , 200:1 by weight ) at room temperature 
 for 0.5 h in the lysis buffer , followed by trypsin digestion ( promega , 
 200:1 by weight ) in 2 m urea , 0.1 m tris - hcl , ph 8.5 at room temperature 
 overnight .
the peptides were then acidified with 0.15% tfa , precleared 
 by centrifugation , desalted with sep - pak c18 spe column ( waters ) , 
 and eluted with 40% acetonitrile ( acn ) plus 0.1%tfa .
protein quantification was carried out by short 
 sds - gel - based staining and bca method .
the desalted 
 peptides from ad brain were resuspended in 10 mm ammonium formate 
 ph 8 at a concentration of 10 mg / ml .
basic ph hplc was performed on 
 a 4.6 mm  250 mm xbridge c18 column ( waters , 3.5 m bead 
 size ) using agilent 1270 hplc instrument .
about 400 g peptides 
 were loaded on the column and hplc gradient started at 90% solvent 
 a ( 10 mm ammonium formate , ph 8.0 ) for 5 min and went up to 50% solvent 
 b ( 90% acetonitrile , 10 mm ammonium formate , ph 8.0 ) during a 50 min 
 time period , followed by a steep increase to 90% b within 5 min at 
 a flow rate of 0.4 ml / min .
the eluted peptides were collected into 
 60 fractions , and every 6 fractions were combined into 10 subfractions 
 in a concatenated pattern to ensure that each subfraction contained 
 similar complexity of hydrophilic and hydrophobic peptides .
the subfractions were then dried and stored at 80 c 
 for further analysis . dried peptides 

peptides were loaded 
 on a 100 m  150 cm column using a nano acquity uhplc 
 ( waters ) system that was interfaced to a q exactive ms ( thermo fisher 
 scientific ) through a nanoelectrospray ion source .
peptides were separated by a designed gradient as indicated 
 ( solvent a : 0.2% formic acid ; solvent b : 70% acn , 0.2% formic acid ) . 

the peak capacity at each gradient time was calculated using formula p = 1 + tg / w , where tg is the time of the gradient 
 and w is the average peak width across entire lc 
 runs .
the peak width of individual lc 
 run was estimated by averaging the chromatographic peak width ( 4 , 
 where 2 is defined as fwhm of the corresponding extracted ion 
 chromatograms ) of major peptide ions .
peptides in the 10 basic ph 
 lc subfractions were resolved similarly on this long column using 
 a 540 min , 1565% buffer b linear gradient .
the q exactive 
 was operated in a data - dependent mode switching between full scan 
 ms and up to 20 ms / ms acquisitions .
the survey scans with an m / z range of 3001600 were acquired 
 in the orbitrap with 35  000 resolution at m / z = 200 and a predicted agc value of 1  10 with maximal ion time of 60 ms .
the ions detected in survey 
 scans were then sequentially isolated and fragmented by hcd at normalized 
 collision energy of 28 ev .
the maximal ion injection time for ms / ms 
 was set to 60 ms at a resolution of 17  500 or 128 ms with a 
 resolution of 35  000 .
different dynamic 
 exclusion times were evaluated to maximize peptide identification 
 including 10 , 20 , 40 , and 60 s. at last , 20 s was chosen for ad brain 
 samples . for gpf method ,
the operation of q exactive ms was similar 
 to the non - gpf method with minor modifications .
the entire m / z range for ms1 was 3001600 but 
 was divided into multiple m / z subsections , 
 which were described in the results and discussion section .
each m / z subsection had 
 10 m / z overlapping with adjacent 
 subsections . for data acquisition of gpf , 

the cycle started at the first m / z subsection of ms1 acquisition , and its data - dependent ms / ms was 
 followed by the second m / z subsection 
 of ms1 acquisition and its data - dependent ms / ms until the full m / z range in ms1 was covered .
the acquired raw ms data 
 were processed with an in - house data - processing pipeline as previously 
 reported .
briefly , the ms raw data were 
 converted to mzxml format using readw software .
up to six precursor 
 ions were selected for a mixed ms / ms spectrum .
the search was performed 
 by the sequest algorithm ( version 28 revision 13 ) against a composite target / decoy human or rat protein database .
the target human protein database was generated from uniprot ( combined 
 swissprot and tremble ) human database containing 71  809 protein 
 entries . the target rat protein database contained 35  570 protein 
 entries .
spectra were searched with 10 ppm for precursor ion 
 mass tolerance ,  0.02 da for fragment ion mass tolerance , fully 
 tryptic restriction , dynamic mass shift for oxidized met ( + 15.9949 ) , 
 two maximal missed cleavages , and three maximal modification sites . 

the peptide spectrum matches 
 ( psms ) were first filtered by the length of matched peptides ( removal 
 of psms with six or fewer amino acids ) and then by mass accuracy . 

the survival psms were further filtered by matching scores to achieve 
 unique protein identification ( grouped using parsimony algorithm ) 
 at 1% fdr . to perform integrative analysis with rnaseq data , we converted 
 uniprot ids to official gene symbols according to ucsc annotation 
 ( downloaded on 01/23/14 ) . for each gene ,
total rna was extracted from 20 
 mg inferior frontal cortex of the same ad brain for proteomics study 
 using the rneasy mini kit ( qiagen ) . on - column 
 dna digestion
the mrna samples were purified by poly(dt ) beads and 
 then fragmented before reverse transcription .
the paired end adaptors 
 were used to ligate the processed double - stranded cdna fragments . 

using bwa ( 0.5.10 ) aligner , rnaseq reads were aligned to 
 multiple databases , including human genome ( grch37 ) , human transcriptome 
 ( refseq and aceview ) , and all possible combinations of refseq exons . 

finally , the reads mapped to the transcriptome were converted to genomic 
 mapping and merged together in the final output bam files .
we packed a 100 m  150 cm nano lc column 
 using 5 m 
 c18 beads and interfaced this column with a q exactive ms for deep 
 shotgun proteomic analysis of mammalian proteome ( figure 1a ) .
recently , the marto group has shown that nano lc columns packed with large beads ( e.g. , 5 
 m ) in extended length ( up to 1 m ) perform as efficiently as 
 nano hplc columns packed with sub 2 m c18 beads for the separation 
 of mammalian protein digest complexes , but the one meter column was 
 operated at a flow rate of 510 nl / min under 1500 psi with 
 a regular hplc system . although the extremely low flow rate may improve 
 ionization sensitivity , it is not optimal for resolving peptides on 
 the majority of nano lc  ms / ms platforms that typically run 
 in the range of 150 to 300 nl / min .
the current 
 long lc system normally flowed at 300 nl / min with backpressure of 
 7500 psi . when heating the column to 60 c with lower flow rate 
 of 150 nl / min , this backpressure was reduced to 3000 psi . 
 to achieve stable electrospray ionization of the eluted peptides , 
 we split the column into two portions ( 110 and 40 cm ) and connected 
 them by a metal zero dead volume union where the voltage was applied .
evaluation 
 of the reproducibility of long lc column coupled to 
 q exactive ms .
( a ) illustration of the setup of long lc column ( 100 
 m  150 cm , 5 m c18 particles ) coupled to q exactive 
 ms .
about 1 g of rat brain tryptic peptide mixture was loaded on 
 the column and then eluted in a 1045% acetonitrile gradient 
 over 4 h. ( c ) comparison of accepted peptide spectrum matches ( psms ) , 
 peptide , and protein identifications . to evaluate the reproducibility of this system
, we examined 
 the 
 run - to - run variation by repeated lc  ms / ms analyses .
the rat 
 brain tryptic peptide mixture was used for the optimization of the 
 system because of similar compositions and dynamic ranges between 
 human and rat brain proteomes . the rat brain peptide mixture ( 1 
 g )
base peak profiles for the replicates were almost identical ( figure 1b ) with the retention time shifts of < 1 min . 

after database search and filtering , the relative standard deviations 
 of accepted psms , unique peptides , and proteins were 2.5 , 2.1 , and 
 0.6% , respectively ( figure 1c ) .
the same column was used for the entire optimization process , comprising 
 more than 100 runs , and no obvious column deterioration was observed .
increasing lc loading 
 capacity is one of the leading approaches to maximize peptide detection 
 in shotgun proteomics analysis .
we examined 
 the effect of peptide loading amount on peptide and protein identifications 
 using this ultralong capillary lc column . when the loading amount 
 of rat brain peptides was increased from 0.2 to 6 g
, the identified 
 peptides and proteins were increased by 60.6 ( from 12  159 to 
 19  529 ) and 39.9% ( from 2105 to 2948 ) , respectively ( figure 2a , 2b ) . however , further 
 addition of loading amount to 20 g resulted in only 1.4% gain 
 of peptides and 0.9% gain of proteins .
consistently , the ion intensities 
 of peptides exemplified by one 1433 peptide and one 
 tbb3 peptide were increased by 10-fold or 3.8-fold , respectively , 
 when the loading amount was increased from 0.6 to 6 g ( figure s1 in the supporting information ) .
however , 
 further improving the loading to 20 g did not lead to stronger 
 ion intensity .
this result suggested that the optimal loading amount 
 of peptides for the current system was 6 g , which was 
 six times higher than the optimal loading amount on a regular capillary 
 lc column ( e.g. , 75 m  12 cm ) and twice as much as the 
 regular loading amount reported on other long lc columns .
various amounts of rat brain peptides were loaded 
 on the long column and analyzed by a 4 h gradient .
( c ) effect of different peptide loading 
 amount on the global distribution of peak width for major peptide 
 ions .
( d ) the effect of loading amounts on the peak width of protein 
 tbb3 peptide nssyfvewipnnvk .
next , we examined the impact of increased peptide loading 
 amount 
 on the lc peak width ( figure 2c ) .
in general , 
 the average peak width increased only 20% ( from 0.65 to 0.77 
 min ) when the loading amount varied from 0.6 to 20 g , indicating 
 that this column has high loading capacity and reasonable performance 
 during chromatography .
interestingly , when loading 20 g of 
 peptides , we found that a fraction of strong peaks showed significantly 
 peak broadening ( figure 2d ) , which may result 
 in ion suppression of adjacent weak peptides .
this observation may 
 also contribute to no gain of identified peptides at 20 g loading . 

to balance the benefit of peak intensity and disadvantage of peak 
 broadening , we selected 6 g peptides as a standard 
 loading level on this lc 
it is desirable 
 to select a lc gradient range in which the number 
 of identified peptides in unit time across the entire lc gradient 
 region is similar to utilize ms instrument efficiently in the shotgun 
 proteomics platform .
we evaluated the 
 lc gradient for the long column and found that the optimal gradient 
 was in a linear gradient range of 1045% acn ( figure 3 ) .
over 98% of the identified peptides were eluted 
 within this gradient range during a 4 h run , and about 78.3  
 21.6 peptides were identified per min .
interestingly , the reported 
 optimal lc gradient range for mammalian cellular tryptic peptide mixture 
 was about 1030% of acn for both regular 12 cm column and long 
 lc column ( up to 50 cm ) .
however , only about half of the 
 peptides were eluted at 30% of acn on this extra - long column , suggesting 
 that higher organic gradient was required for efficient elution of 
 mammalian cellular peptide complex on ultralong c18 lc columns .
this observation may be explained by the increasing 
 interaction between peptides and c18 beads created by the long distance 
 through which peptides have to travel .
g of peptides was loaded on the long column and eluted in a 
 1045% gradient of acetonitrile over 4 h. the lc elution profile 
 was represented by total ion current ( solid black line ) along with 
 the gradient ( dotted black line ) .
the number of identified peptides 
 every 2 min was plotted ( solid red line ) .
about 157  42 peptides 
 were identified in every two min . to determine the optimal gradient time for peptide and protein 
 identifications on this system ,
we tested various lc gradients ranging 
 from 2 to 12 h. we first calculated the average peptide peak width 
 of major peptide ions across the entire elution and then derived the 
 corresponding peak capacity for each gradient ( figure 4a ) . when the peak capacities were plotted as a function of 
 gradient time , a positive correlation was observed between peak capacity 
 and gradient time .
the peak capacity reached its maximum of 730 , similar 
 to the reported peak capacities of other nano lc columns used for 
 in - depth proteomics analysis .
next , we investigated 
 whether the increased peak capacities can lead to more peptide identifications . 

as expected , the number of psms was increased proportionally to the 
 extended gradient time , while the number of identified peptides and 
 proteins also followed this trend ( figure 4b ) .
the number of detected peptides and proteins almost reached plateau 
 at 12 h gradient with the identification of 23  884 peptides 
 and 3484 proteins from 46  711 psms .
interestingly , there was 
 a linear correlation ( r = 0.985 ) between 
 the peak capacity and the number of identified peptides ( figure 4c ) , supporting the notion that peak capacity is 
 a major factor for optimizing lc  ms / ms - based peptide identification .
peak capacities were calculated 
 by dividing the average peak width of major peptide ions in a lc run 
 over entire gradient time .
( b ) correlation between the number of identified 
 peptides / proteins and gradient time .
( c ) number of detected peptides 
 was in a linear relationship with the peak capacity .
one 
 interesting finding 
 was that ms sequencing efficiency was reduced when lc gradient time 
 was extended on the long column , evidenced by a steady decline of 
 the ratios of summed ms2 scans versus ms1 scans ( figure s2a in the supporting information ) .
this result suggested 
 that there was not a sufficient number of ions detected in survey 
 ms1 scans to trigger ms2 scans . because gpf is capable of detecting 
 weak sample ions within a narrow m / z range
but it takes multiple ms1 scans to cover a full scan region , we assessed the function of gpf to improve the ms sequencing efficiency . 

the m / z subsections of gpf were 
 determined experimentally to contain the same number of psms in each 
 subsection of m / z windows using 
 rat brain peptides as a testing sample . during a 4 h lc 
ms / ms 
 analysis , one , two , three , and four m / z subsections in a full m / z range 
 of ms1 were tested ( table s1 in the supporting 
 information ) . compared with no gpf ,
the implementation of gpf 
 of three subsections exhibited the highest ratio of ms2/ms1 scans 
 ( figure s2b in the supporting information ) and led to 11.3 and 15.4% increase in the number of identified 
 peptides and proteins , respectively ( figure s2c 
 in the supporting information ) .
thus , the gfp of three subsections 
 was chosen for later experiments . to further optimize the sequencing 
 efficiency of ms
, we evaluated the effect of different dynamic exclusion 
 time of ms on the identification of peptides and proteins .
in a 4 
 h lc gradient on the long lc column , the number of psms , peptides , 
 and proteins was the highest at 20 s dynamic exclusion time ( figure s3 in the supporting information ) .
because 
 the calculated average peak width was 40 s for the 4 h lc 
 gradient , each m / z ion would be 
 analyzed about twice .
reduction of the dynamic exclusion time from 
 20 to 10 s leads to 22 and 15% drop in the number of peptide and protein 
 identifications , respectively , even though the ms2/ms1 ratio reached 
 the highest number of 10 .
this result clearly showed the redundant 
 sampling at 10 s dynamic exclusion time because of repetitive sequencing 
 of the same peptide ions .
we observed 1.9% decrease in the number 
 of peptide and protein identifications and > 23% dropping of psms 
 at 
 40 s dynamic exclusion time .
therefore , the dynamic exclusion time 
 was set at 20 s for the 4 h lc gradient .
we then used the 
 optimized conditions to explore the entire proteome of ad brain ( figure 5a ) .
about 400 g of protein was extracted 
 from the tissue and subjected to lys - c and trypsin digestion .
we digested 
 the entire cell lysate without the removal of cell debris to increase 
 the coverage of membrane and nucleus proteins as a recent study suggested .
basic ph reverse - phase lc was performed to prefractionate 
 ad brain peptides because it provides better resolution and loading 
 capacity than other methods ( e.g. , scx , hilic ) and good orthogonality 
 to acidic reverse - phase lc ( figure 5b ) .
we collected 10 basic ph lc fractions and analyzed each fraction 
 on the acidic ph lc 
total 
 ms instrument time was 4 days , which is comparable to other 
 reported instrument times required for in - depth proteomes analysis 
 varying from 1 to 2 weeks .
a total of 1  695  626 high - resolution ms / ms spectra 
 were acquired , identifying 629  747 psms ( 37% successful rate ) , 
 96  127 peptides , and 10  544 proteins when protein fdr 
 was controlled at 1% . on average ,
each peptide was identified by ms 
 for about seven times . in each basic ph lc fraction , the average number 
 of detected peptides and proteins were 119  303  651 and 
 4701  119 , respectively ( figure 5c ) . 

nearly 80% of peptides were solely identified in one fraction and 
 95% of peptides were only found in one or two fractions , suggesting 
 high partitioning of peptides within each fraction ( figure 5d ) .
( b ) chromatograph of basic ph rplc prefractionation of 
 peptides ( upper panel ) monitored at 214 nm and an example base peak 
 chromatograph of acidic ph long gradient rplc 
ms / ms ( lower 
 panel).(c ) basic ph rplc fractionation yielded even partitioning of 
 peptides , which led to similar number of identified proteins in concaternated , 
 pooled fractions .
to evaluate the depth 
 of ad brain proteome analyzed in this pilot 
 study , we performed deep rna - seq analysis of the same sample and compared 
 the proteome data with transcriptome results .
we identified 16  670 
 protein coding genes by rna expression , similar to the result in previous 
 transcriptomic analysis of human brain .
the abundance of each transcript was calculated as reads in fragment 
 per kilobase of exon per million fragments mapped ( fpkm ) . a total 
 of 10  161 human genes
were detected in ad brain proteome , corresponding 
 to 61% of the expressed genes ( figure 6a ) . 

next , we investigated the correlation between transcript and protein 
 levels in our study .
the protein level was indicated by a spectral 
 counting based method , in which the total 
 number of spectral counts for every protein was summed and normalized 
 by the length of protein sequence ( spectral counts per thousand amino 
 acids ) to adjust the bias created by protein size .
we observed a modest 
 correlation between the rna and protein levels ( spearman correlation 
 = 0.62 , figure 6b ) , which was consistent with 
 the conclusions of other studies ( spearman correlation = 0.4 to 0.6 ) .
taken together , our data suggested that the 
 utilization of the current optimized lc / lc  ms / ms platform covers 
 the majority of ad brain proteome .
comparison of deep proteomics and rna - seq 
 data from the same ad 
 brain tissue .
( a ) histogram of fpkm distribution of rna - seq and proteomics 
 data .
the open bar represents the distribution of protein coding gene 
 numbers detected by rnaseq , and the gray bar indicates the distribution 
 of protein coding gene numbers validated by ms with different fpkm 
 values .
( b ) scatter plot of spectra counts per thousand amino acid 
 of proteomic data versus fpkm of rna - seq data .
ms / ms system presented here 
 was 
 robust with no instrument down time during the entire process of deep 
 proteomic analysis . by heating the lc column to 60 c , this lc 
ms / ms 
 system can be operated under regular pressure limit ( 3000 
 psi with 0.15 l / min flow rate ) , reducing potential problem 
 of overpressure .
it is conceivable that extensive prefractionation 
 of peptides during basic ph lc separation would further reduce sample 
 complexity and improve dynamic range in the prefractionated pools 
 to further enhance the identification of extremely low - abundance proteins . 

at last , the combination of our long column lc / lc  ms / ms platform 
 with the newly introduced orbitrap fusion tribrid mass spectrometer 
 would also allow considerably deeper proteomics analysis due to its 
 higher scan rate and peptide identification efficiency than the q 
 exactive ms instrument used in this study .
we have demonstrated a reverse - phase - based , 
 multidimensional long 
 gradient lc  ms / ms platform suitable for deep proteomics analysis . 

we systematically examined and optimized various parameters of a 100 
 m  150 cm lc column packed with 5 m reverse - phase 
 c18 beads .
the column exhibits great robustness and reproducibility 
 together with high peak capacity ( 700 ) and loading capacity 
 ( optimal at 6 g ) . using this column in conjunction with basic 
 ph lc and q exactive ms with gpf ,
the identification of a deep proteome 
 of ad brain ( > 10  000 proteins ) was achieved in 4 
 days 
 of ms instrument time .
 the 
 development of high - resolution liquid chromatography ( lc ) is 
 essential for improving the sensitivity and throughput of mass spectrometry 
 ( ms)-based proteomics . here 
 we present systematic optimization of 
 a long gradient lc  ms / ms platform to enhance protein identification 
 from a complex mixture . the platform employed an in - house fabricated , 
 reverse - phase long column ( 100 m  150 cm , 5 m 
 c18 beads ) coupled to q exactive ms . 
 the column was capable of achieving 
 a peak capacity of 700 in a 720 min gradient of 1045% 
 acetonitrile . 
 the optimal loading level was 6 g of 
 peptides , although the column allowed loading as many as 20 g . 
 
 gas - phase fractionation of peptide ions further increased the number 
 of peptide identification by 10% . 
 moreover , the combination 
 of basic ph lc prefractionation with the long gradient lc  ms / ms 
 platform enabled the identification of 96  127 peptides and 
 10  544 proteins at 1% protein false discovery rate in a post - mortem 
 brain sample of alzheimer s disease . because deep rna sequencing 
 of the same specimen suggested that 16  000 genes 
 were 
 expressed , the current analysis covered more than 60% of the expressed 
 proteome . further improvement strategies of the lc / lc  ms / ms 
 platform were also discussed . 
Introduction
Experimental Procedures
Results
and Discussion
Conclusions