as genomes have been sequenced and sequence databases have 
 been compiled , these databases have been used to identify proteins 
 present in biological samples using tandem mass spectrometry data . 
 in a process called  shotgun proteomics  , mixtures of 
 proteins from biological samples are digested to peptides using proteases , 
 and liquid chromatography coupled to tandem mass spectrometry is used 
 to directly identify the peptides present .
identification 
 of a peptide is used to infer the presence of the protein it is derived 
 from . as more complex samples , such as cellular components and whole 
 cells , have become targets of analysis , more comprehensive separations 
 have become necessary to resolve complex peptide mixtures and increase 
 dynamic range .
giddings described multidimensional 
 chromatography as a means to increase peak capacity by combining orthogonal 
 separations .
for the analysis of complex 
 peptide mixtures , several combinations of separation media have been 
 used . the last phase in multidimensional liquid chromatography ( mdlc ) 
 separations used for mass spectrometry is typically reversed - phase 
 ( rp ) , which separates peptides by hydrophobicity and is effective 
 at removing salts or other small molecule contaminants prior to introduction 
 of peptides into the mass spectrometer .
many different forms of mdlc have appeared over the years , from 
 combinations of ion exchange ( iex ) with rp separations to combinations 
 of capillary electrophoresis and liquid chromatography . in any multidimensional separation ,
how material is transferred 
 from one separation stage to another is critical for maximizing peak 
 capacity and optimizing sample recovery . in proteomics ,
link et al . employed 
 a biphasic column of strong cation exchange ( scx ) and rp packing material 
 in a single column . in this arrangement , a multidimensional separation 
 is created by running buffer containing a set concentration of salt 
 across the column to elute peptides from the scx phase onto the rp 
 column .
once the salt pulse is completed , the rp buffer is applied 
 to the column at 0% b to remove salt from the column prior to running 
 the gradient . after a rp gradient
is completed , a second salt pulse 
 with a higher concentration of salt is applied to the column to move 
 a new population of peptides to the rp material . the process of salt 
 pulse / rp gradient is repeated until all peptides are removed from 
 the scx phase .
a single column contains both the iex phase and the rp phase , and 
 all solvents flow over both phases .
the sample is loaded directly 
 onto the column from an eppendorf tube using a pressurized device , 
 and the column is placed in line with the ion source with the voltage 
 placed on a waste line at the backend of the column .
this method is commonly referred to as online mdlc and may employ 
 a bi- or triphasic column .
this method usually employs scx , but strong anion exchange 
 ( sax ) has also been used .
advantages 
 to off - line fractionation include the ability to add a high organic 
 phase to the salt buffer ( e.g. , 25% organic in the iex buffer ) to 
 minimize mixed - mode interactions , the capability to collect many fractions , 
 and the capacity to load large amounts of material onto the column . 
 in a clever use of off - line fractionation , wang et al .
showed improvement 
 in peptide identifications by combining fractions from different parts 
 of a high ph rp separation to produce collections of peptides with 
 different physical characteristics like hydrophobicity for the second - dimension , 
 low - ph rp separation .
after rp separation , excess solvent 
 in the collected fractions is removed , and each fraction is loaded 
 into an autosampler for introduction into the mass spectrometer .
a 
 third strategy for multidimensional separation is also an online method 
 that employs a valving system to direct solvents to an iex column , 
 an enrichment column , a rp column , and waste .
this system represents 
 a compromise between the direct online and the off - line approaches . 

the valving system is used to redirect flow to shunt salt solutions 
 used to elute peptides from an iex column to waste rather than have 
 it run through the rp analytical column , or in the case of a rp - rp 
 lclc system , the valves are used to direct peptides to the enrichment 
 column to alter the ph of the buffer before the analytical separation .
sample loading is a critical part of capillary chromatography , 
 as these systems involve small diameter openings that must be aligned 
 and low solvent flows , for which dead space can have a great impact . 

kennedy and jorgenson developed a pressurized device for both packing 
 and loading capillary columns .
the end 
 of the column was placed directly into a slurry of packing material 
 and when the device was pressurized the packing material was driven 
 into the column .
this same strategy could be used as a means to load 
 samples directly from eppendorf tubes into a column .
this method has 
 been adopted by others as a means to load small quantities of samples .
an important consideration when analyzing 
 peptides and proteins 
 is sample loss associated with sample handling . proteins and peptides 
 can easily adhere to surfaces resulting in losses . a carrier protein 

is often used to minimize adherence to active surfaces during sample 
 manipulations to protect low abundance proteins from losses .
an advantage of shotgun proteomics is the manipulation 
 of complex protein mixtures where the more abundant proteins may presumably 
 act as carrier proteins to protect lower abundance proteins from loss . 
 because losses can occur on active surfaces such a glass and metal , 
 efforts
two recent papers showed that peptides can be 
 lost when analyzed from autosamplers , and peptide mixtures of intermediate 
 complexity ( in gel digestions ) can be lost to the surface of autosampler 
 vials made from a variety of materials , respectively .
tested a variety of carriers to determine which 
 one improved recovery of peptides . as 
 these two papers have shown , and as experience has taught us , the 
 more samples are handled and exposed to surfaces , the greater the 
 loss .
this study compares sample losses for a shotgun proteomics experiment 
 using three different methods of sample introduction using two different 
 quantitation methods .
hek293 cells ( crl-1573 ) 
 purchased from atcc were seeded into a t25 flask in supplemented media 
 ( dmem medium supplemented with 10% fetal bovine serum , 1% ( v / v ) penicillin / streptomycin , 
 2 mm l - glutamine , and 200 g / ml g418 ) from ( gibco , 
 invitrogen)),and maintained with regular media changes for 3 weeks 
 before they were considered to be stable cell lines .
cultured cells were harvested at 80% confluency 
 with 0.05% trypsin and edta , centrifuged for 5 min at 4000 g at 4 c , and washed twice with pbs .
cells were suspended 
 in 8 m urea , 500 mm tris - hcl ph 8.5 supplemented with complete ultra 
 tablets , mini , easypack ( roche , mannheim ) for protein extraction .
denaturated protein lysate was 
 precipitated with acetone and assayed using modified bicinchoninic 
 ( bca ) method ( pierce , rockford il ) .
resuspended 
 protein was reduced with 5 mm tris(2-carboxyethyl)phosphine ( tcep ) 
 for 30 min .
cysteine residues were alkylated with 10 mm iodoacetamide 
 for 20 min in the dark .
samples were diluted 
 to a final concentration 2 m urea with 100 mm tris - hcl , ph 8.5 prior 
 to digestion with trypsin . for endopeptidase digestion ,
modified trypsin 
 ( promega , madison , wi ) was added at 50:1 ( protein / protease mass ratio ) 
 along with 1 mm cacl2 and incubated overnight in a thermoshaker 
 at 600 rpm at 37 c .
digested peptide solution was acidified 
 using 90% fa to a final ph of 3.0 .
an analytical rplc column was prepared by pulling a 100 
 m id/360 m od capillary ( polymicro technologies , phoenix , 
 az ) to 5 m i d tip .
reversed - phase resin ( aqua c18 , 3 m 
 dia . , 90  pores , phenomenex , torrance , ca ) was packed directly 
 into the pulled column at 700 psi until 12 cm long .
the column was 
 washed and equilibrated at 100 bar with buffer b , followed by buffer 
 a. a multiprotein identification technology 
 ( mudpit ) 
 trapping column was prepared by creating a kasil frit at one end of 
 an undeactivated 250 m id/360 m od capillary ( agilent 
 technologies , santa clara , ca ) .
the frit was prepared by briefly dipping 
 a 20 cm capillary in well - mixed 300 l of kasil 1624 ( pq corporation , 
 malvern , pa ) and 100 l of formamide , curing at 100 c 
 overnight , and cutting the frit to 1.5 mm in length .
triphasic or biphasic columns were successively packed with 2.5 cm 
 scx particles ( partisphere scx , 5 m dia .
, 100  pores , 
 phenomenex ) and 2.5 cm rp resin ( aqua c18 , 3 m dia . , 125  
 pores , phenomenex ) , as shown in figure 1 .
peptide 
 samples ( 100 g ) were loaded onto triphasic columns 
 for online mudpit .
for offline mudpit , samples were loaded onto a 
 biphasic column and ten scx offline fractions were collected in 1.5 
 ml eppendorf tubes .
fractions were then loaded into a 2.5 rp resin 
 column ( offline mudpit ) or purified by stage tip and placed in autosampler 
 vials ( offline mudpit with autosampler [ easy - nlc ii , thermo ] ) .
both 
 mudpit and analytical columns were assembled using a zero - dead volume 
 union ( upchurch scientific , oak harbor , wa ) .
( a ) hek293 protein lysate was digested and processed in three replicate 
 runs with different mudpit panels : online ( automated panel ) , offline 
 ( manual collection of fractions ) , and offline - as ( offline with autosampler 
 where fractions were collected manually then cleaned up with c18 stage 
 tip columns before being placed into autosampler ) .
( b ) offline fractions 
 were collected for 5 min after the volatile salt pulse phase using 
 10100% ammonium acetate .
sixplex tandem mass 
 tag ( tmt ) labeling was performed according 
 to the manufacturer s instructions ( thermo fisher scientific , 
 rockford , il ) .
as illustrated in figure 2 , 
 tmt reagents ( 0.8 mg ) were dissolved in 40 l of anhydrous acn 
 ( sigma , milwaukee ) .
trypsin - digested hek293 cells samples ( 25 g / tag ) 
 were equilibrated to room temperature for 5 min with occasional vortexing . 

samples were then resuspended in 100 mm teab and derivatized with 
 sixplex chemical tags : 126 , 127 for online mudpit ; 128 , 129 for offline 
 mudpit ; and 130 and 131 th ( thomson ) for offline mudpit with autosampler . 

the reaction mixtures were incubated at room temperature for 1 h and 
 quenched with 15 l of 5% hydroxylamine solution in water .
equal 
 ratios of tmt- tagged samples were mixed and analyzed prior to fractionation 
 to ensure unbiased and impartial labeling .
fractions were massed up 
 to same volume with 100 mm teab and equally combined into one sample 
 before vacuum drying .
the lyophilized tmt - labeled peptides ( 25 g ) 
 were reconstituted with 50 l of buffer a ( 0.1% formic acid 
 ( fa ) , 5% acetonitrile ( acn ) in water ) , centrifuged at 12  000 g for 30 min prior to mass spectrometric analysis .
protein lysate was labeled 
 in a sixplex tmt format : 126 and 127 for online mudpit , 128 and 129 
 for the offline mudpit , and 130 and 131 for the offline with autosampler 
 mudpit ( offline - as ) . directly after labeling ,
an equal volume of the 
 tmt tags was mixed and analyzed to ensure successful labeling ( labeling 
 efficiency averaged 97.2% ) .
samples were then processed with different 
 mudpit platforms , massed up to same volume with 100 mm teab , and equally 
 combined into one sample before drying and mass analysis .
peptides 
 were separated by 
 an eksigent nanolc-2d system ( eksigent , dublin ) with or without autosampler 
 unit ( 10 l peek sample loop , six - port titanium injection valve , 
 50 mm sus sample needle , 50 m i d fused silica tubing ) .
the 
 hplc system was either connected online or offline to thermo ltq xl 
 ( for label - free quantification ) or ltq - orbitrap velos ( for tmt quantification ) 
 using an in - house built nanoelectrospray stage .
electrospray was performed 
 directly from the analytical column by applying the esi voltage at 
 a tee ( 150 m i d , upchurch scientific ) directly downstream of 
 a 1:1000 split flow used to reduce the flow rate to 250 nl / min through 
 the columns .
ten - step mudpit experiments 
 were performed either online or offline , with steps corresponding 
 to 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , and 100% buffer c being run 
 for 5 min at the beginning of a 120 min gradient .
a three mobile phase 
 system consisting of buffer a ( 5% acn ; 0.1% fa ( sigma - aldrich , st . 

louis , mo ) ) , buffer b ( 80% acn , 0.1% fa ) , and buffer c ( 500 mm ammonium 
 acetate , 5% acn , 0.1% fa ) was used in the current experiment .
the 
 lc system was coupled to 224 nm laser - induced native fluorescence 
 ( linf ) detector with elliptical flow cell for real - time peptide detection .
data - dependent acquisition of ms / ms spectra 
 was performed by dynamically choosing up to 5 or 10 most intense precursor 
 ions from the survey scan for ltq xl or ltq orbitrap velos , respectively . 

, 
 charge  25 , full - scan ms resolution of 30.000 ( ltq 
 xl ) and 60.000 ( ltq orbitrap velos ) with a target value of 1  
 10 , and the maximal injection time of 200 ms .
the lower 
 threshold for targeting a precursor ion in the ms scan was 5000 counts 
 and 2.5 kv maximum injection time for higher - energy collisional dissociation 
 ( hcd)ms / ms analysis in the orbitrap .
the hcd dissociation 
 mode enables simultaneous production of tmt reporter ions and fragment 
 ions of the peptides .
ms / ms scans were 
 acquired in the orbitrap with a mass resolution of 17  000 . 

the target value was 30  000 ions with injection time of 150 
 ms .
once analyzed , the selected peptide ions were dynamically excluded 
 from further analysis for 120 s to allow for the selection of lower - abundance 
 ions for subsequent fragmentation and detection using the setting 
 for repeat count = 1 , repeat duration = 30 ms , and exclusion list 
 size = 500 . ions with singly or unassigned charge states were rejected . 

the m / z isolation width for ms / ms fragmentation was set to 2 th . 
 for
tandem mass spectra were extracted from 
 raw files using rawextract 1.9.9 and 
 searched with the prolucid algorithm against homo sapiens uniprot / swiss - prot database with reversed sequences 
 ( 176  708 entries ) .
the search space included all fully and 
 semitryptic peptide candidates ( at least six amino acids ) .
carbamidomethylation 
 of cysteine ( 57.02146 amu ) was considered as a static modification 
 as well as static n - terminus and lysine modification ( 229.1629 amu ) 
 for sixplex tmt labels analysis .
the 
 search parameters include 10 ppm precursor mass tolerance and 0.6 
 da peptide mass tolerance
. exported prolucid files were assembled 
 and filtered using the dtaselect2.0 which combines xcorr and deltacn 
 values using a quadratic discriminate function to compute a confidence 
 score .
the false - positive rate ( fdr ) 
 was kept at 1% at the protein level . for quantitative analysis ,
census 
 was used to extract the relative intensities of reporter ions for 
 each peptide from the identified tandem mass spectra for normalization . the mass tolerance and intensity threshold for 
 the reporter ions in census
prolucid xcorr , deltacn , and zscore values were used 
 to generate a bayesian discriminator .
outlier points in the two distributions 
 that had a mahalanobis distance greater than four were discarded . 
 for label - free quantification , normalized spectral abundance factor , 
 protein , peptide expression alteration ( fold changes ) , log values , 
 and confidence were calculated based on spectral peak intensities 
 generated from the mass spectrometric analysis after extracting confident 
 protein spectra with p < 0.01 . for tmt analysis , 

the relative quantification between any experimental groups in the 
 sixplex experiment was derived from the average ratio of the reporter 
 ions of duplicate tags of one group over the average reporter ions 
 of duplicate tags of the corresponding group .
statistical computing 
 and graphics were performed in r software environment and graphpad 
 prism 5 .
although the use of capillary chromatography has provided tremendous 
 gains in sensitivity , the use of capillary chromatography also introduces 
 sample - loading challenges .
kennedy and jorgenson introduced a loading 
 procedure that used a high - pressure device (  bomb  ) 
 to transfer samples directly from an eppendorf tube into the capillary 
 column .
when the bomb is pressurized , 
 liquid is forced into the column from the eppendorf tube .
the capability 
 to analyze small amounts of materials allows access to samples such 
 as biopsy samples , small sections of tissue such as brain sections , 
 or even single - cell analysis such as neurons . as demonstrated by masuda 
 et al . and
thakur et al , as sample size decreases , sample handling 
 and the nature of the chromatographic interface become important for 
 good detection limits on peptides .
in this study , 
 we tested three methods for introducing complex peptide mixtures into 
 a tandem mass spectrometer .
the three multidimensional lc methods 
 consisted of a direct online method using an integrated triphasic 
 capillary column for the introduction of samples and two off - line 
 methods involving collection of samples in eppendorf tubes .
one of 
 the off - line methods used an autosampler to introduce the samples 
 into the analytical rp column , and the other used the same direct 
 pressure loading system used to load samples for the online mdlc analysis . 

, all approaches used the same type of scx column 
 to perform the iex fractionation , which consisted of a biphasic column . 

in addition , 10 salt steps were used in the online system , and 10 
 fractions were collected in the two off - line methods using the same 
 elution method . the same analytical rp method was employed . to perform 
 the comparison ,
a trypsin - digested hek293 sample was used that should 
 be representative of the type of sample commonly analyzed in proteomics . 
 for each method ,
two different quantitation methods were 
 used to measure differences in peptide recoveries between the different 
 methods . even with the current advanced mass spectrometric capabilities , 
 multiple dimensions of separation improve comprehensive analysis of 
 peptide mixtures .
although a variety of multidimensional combinations 
 have emerged in the past few years , the scx - rp combination is an efficient and highly resolving separation 
 method for shotgun proteome analysis .
the main impetus of this work 
 is to provide a systematic comparative study of different lc / lc strategies 
 in terms of performance , sensitivity , and recovery that should be 
 applicable to all lc / lc methods regardless of the phases employed proteins from 
 a hek293 cell line were digested with trypsin and aliquots 
 were subjected to analysis by the three different platforms to measure 
 peptide and protein identification efficiency for each method . for 
 this measurement ,
the goal was simply to report how many peptides 
 and proteins would be detected for the same sample using the different 
 methods ( figure 1 ) . because of the complexity 
 of the sample , 10 salt steps were used to ensure elution of most peptides 
 from the column .
three replicate analyses of each platform showed 
 an average of 187  366  8545 , 168  750  3113 , 
 and 140  000  7950 ms2 scans from online , offline , and 
 offline - as groups , respectively .
a total of 3383  386 , 2159  243 , and 1877 
  413 proteins were identified from the 12937  1533 , 7351 
  1201 , and 7146  829 peptides identified for the online , 
 offline , and offline - as groups , respectively ( figure 3a , b ) .
the higher mean of the online triphasic column was statistically 
 significant ( confidence of 95% ) compared with the other methods .
given 
 the identical gradients and ms methods used for each approach , the 
 peptide identification and protein identification numbers were similar 
 to an improvement by more than 1.7 to 1.8 fold in the online method 
 compared with both offline methods ( figure 3b ) .
additionally , merging and removing redundant proteins / peptides 
 increased protein / peptide identifications most in the online triphasic 
 column method , less in the offline method , and least in the offline - as 
 method .
this comparison showed 
 that recovery of peptides was best in the online method by virtue 
 of the most peptide and protein identifications and the poorest in 
 the off - line fractionation method with the introduction of samples 
 through an autosampler .
this result makes sense , as the samples being 
 fractionated offline and introduced through an autosampler are being 
 exposed to more new surfaces and are being subjected to the most manipulations .
box and 
 whiskers plots of averaged identified proteins ( a ) and 
 peptides ( b ) for different mudpit platforms .
a key measure in proteomics is overlap in peptide and protein identifications 
 as a function of technical replicates .
reproducibility between protein 
 identifications is expected to be greater because a protein can be 
 identified by different peptides .
a high level of reproducibility 
 in peptide identification is harder to achieve because it requires 
 the system be near saturation .
a comparison of reproducibility and 
 overlap between the different systems is shown in figure 4 .
proportional venn diagram demonstrating the overlap between 
 protein 
 identification of different mudpit triplicate runs ( ( a ) online , ( b ) 
 offline ; and ( c ) offline - as ) or between different mudpit platforms 
 ( d ) .
reproducibility between runs was 
 greatest among online replicates 
 ( 60% overlap ) and lowest in the offline analysis coupled to the autosampler 
 ( 45% overlap ) .
in addition , a comparison of the three experimental 
 strategies shows online lc / lc identifies more distinct proteins at 
 1% fdr compared with the other platforms ( figure 4d ) .
differences in identification rates among the samples 
 suggest that improved identification rates are a result of minimizing 
 manual handling of fractions . by using an online system sample ,
losses 
 are decreased , and this leads to improved recovery of peptides through 
 the system and acquisition of more ms / ms . if there is an observed 
 difference in peptide identification between the different methods , 
 it begs the question of whether there are any differences in physicochemical 
 characteristics of the peptides or proteins observed in the different 
 methods .
we analyzed the proteome metrics relevant to each experimental 
 platform to determine if there were any peculiar physicochemical characteristics 
 of peptides observed in one platform and not the other .
in general , 
 online mudpit showed only a modest increase in protein sequence coverage 
 ( figure 5a ) over the offline methods .
spectral 
 count rank , for example , abundance of proteins , however , was significantly 
 lower in the offline - as platform compared with the other methods ( figure 5b ) . because the major difference in the offline - as 
 group is that the final peptide mixture is placed in autosampler vials , 
 it is likely that the lower spectral counts are due to adsorptive 
 loses of analyte on the surface of the polypropylene sample vials or that sample is lost in the flow path of the sample loop .
peptide loss in the offline groups did not significantly 
 correlate with pi , salt fraction , or peptide charge 
 ( figure 5c  e ) .
although we included 
 organic modifier ( 5% v / v acn ) in the iex elution buffer that reportedly 
 reduces surface adsorption , our data 
 suggest a modest loss of hydrophilic proteins in the offline - as group 
 when plotted using bull breese or kyte  doolittle scores ( figure 5f , g ) .
this finding is in accordance with recent 
 reports , describing a higher adsorptive tendency of soluble peptides 
 to solid surfaces that ultimately affects peptide amount and quantification 
 parameters .
( d ) number of peptides eluted 
 in each salt fraction of the mudpit platforms .
( f ) bull breese hydrophobicity index was calculated 
 based on the free energy of transfer to surface in kcl / mol .
( g ) kyte  dolittle 
 hydrophathy scoring ( gravy score ) was calculated based on the average 
 amino acids score for a given protein .
positive score is hydrophilic 
 and negative score is hydrophobic in bull breese and vice versa for 
 kyte  dolittle .
red is online mudpit , blue is offline mudpit , 
 and black is offline - as mudpit . to further illustrate the changes in protein abundance 
 between groups
, we performed a statistical comparison of the average 
 spectral count of triplicate runs from each experimental platform . 
 as illustrated in figure 6 , although several 
 proteins were quantified in high abundance between all platforms , 
 we noticed that online proteins were more abundant and statistically 
 more significant ( p  0.01 ) , especially when 
 compared with either of the offline groups ( offline or offline - as ) . 

this significance was less obvious when comparing between the offline 
 groups ( figure 6c ) .
specifically , we found 
 that 635 and 542 proteins were significantly higher 
 in abundance in the online platform compared with offline and offline - as 
 groups by 2.2  0.44 and 1.8  0.52 fold , respectively .
label - free 
 quantification based on normalized spectral abundance 
 counts of different mudpit panels .
( a  c ) volcano plots depict 
 relationship between the p value and the magnitude 
 of difference ( log2 ) in expression value between average 
 technical replicates of two compared groups .
( d  f ) surface 
 3d plots of the identified proteins in each experimental group based 
 on their relative spectral counts ( % ) , molecular weight ( kd ) , and 
 protein length . to verify our 
 findings with an alternate quantitation method
, peptides were labeled 
 with different amine - reactive isobaric tags ( figure 2 ) .
the tmt experiment was designed as another way to quantitate 
 the differences between the strategies for lc / lc . a digested hek293 
 sample was aliquoted into six aliquots of 25 g each .
two tagged samples each were 
 used for online separation : two for off - line with pressure bomb loading 
 and two for off - line fractionation with autosampling .
the experiments 
 were performed as for the label - free experiments except that the outflow 
 from the rp capillary column was collected into a single tube for 
 each sample .
the volumes were adjusted ; then , all samples were combined 
 into a single tube .
this method ensures exact comigration with simultaneous 
 and accurate peptide quantification using the mass tags that appears 
 in the tandem mass spectrum .
we found 
 that peptide abundance was lower ( p  0.05 ) 
 in the offline groups ( figure 7a  c ) . 

again , this was not restricted to certain peptides properties ; in 
 contrast , the majority of them showed similar trend patterns , denoting 
 stochastic nonspecific loss .
in addition , peptide ratios between online 
 and offline groups disclosed a modest skew toward the online platform 
 with a 1318% increase after peptide grouping and normalization 
 ( figure 7d ) .
cumulatively , the elevated ion 
 intensity signaling for peptides detected in the automated online 
 method corresponded to an average increase of 18% in protein abundance 
 for more than 1100 proteins ( figure 7e ) .
we 
 noticed that the same proteins were underestimated in the offline 
 groups with a significant correlation coefficient factor ( r = 0.76 , p = 0.05 ) when compared with 
 the online platform ( figure 7e ) .
( b ) peptides were plotted with trend line pattern ( dashed red line ) . 

( c ) perpendicular 3d plot revealed that most of the identified reporter 
 ions were relatively higher in online mudpit with respect to the other 
 offline formats .
( d ) frequency histogram of mudpit panels showing 
 distribution of log2 peptide ratio observed between compared 
 groups .
( e ) log  log correlation plot of protein expression 
 ratio of the online panel over offline panels .
black and red dots 
 represents proteins with higher intensities in online module with 
 1.4 to 2 fold , respectively .
( f ) example spectrum for peptide labeled 
 with tmt isobaric mass tag labeling reagent .
the ms / ms fragmentations 
 were used to sequence the peptide . on the basis of the amino acid 
 ladder ,
the peptide was identified as vnptvffdiavdgeplgr 
 with the n - terminus modified by tmt isobaric mass tag labeling reagent . 

mass tags 
 ( 126131 ) observed in the lower m / z region ( inserted figure ) indicate the relative abundance 
 of this peptide in each group .
the samples were labeled in the following 
 order : online mudpit ( 126 , 127 ) , offline mudpit ( 128 , 129 ) , and offline 
 mudpit with autosampler ( 130 , 131 ) . to answer the question of whether the lowered peptide intensity 
 in the offline methods is due to peptide loss during processing or 
 ion suppression as a result of high background noise ( chemical contaminants , 
 atmospheric sources , or electrical interference )
, we utilized the 
 high sensitivity of the linf detector ( 100 fold than uv detectors ) together with peptide quantification using modified 
 bca method .
peptide mapping 
 at 220 nm provided a flat baseline with better sensitivity in the 
 current experiment ( figure 8a ) , and detection 
 of scx eluted peptides before mudpit processing did not show any significant 
 differences between experimental groups ( figure 8b ) even at 280 nm associated with absorption of the aromatic amino 
 acids ( data not shown ) . nevertheless , downstream peptide quantification , 
 directly before mass spectrometry analysis , revealed potential low 
 peptide yield ( p  0.05 ) in the offline methods 
 ( figure 8c ) .
a previous report 
 demonstrated that the adsorption of biomolecules such as peptides 
 followed a langmuir isotherm equation and was influenced by both solvents 
 and the nature of the solid surfaces .
moreover , we monitored the possible impact 
 of sparse ion noise background on the desired peptide signal peak 
 by calculating signal - to - noise ratio ( s / n ; figure 8d ) based on gygi s method .
although most peptides in our analysis were analyzed at low s / n , 
 the consequences of low signal levels on quantitative accuracy remain 
 to be tested .
interestingly , online mudpit generated slightly higher 
 background noise compared with offline and offline - as ; the s / n ratio , 
 however , was not significantly different between all platforms ( figure 8d ) , and both ms and ms / ms quality was comparable . 
 this observation could be attributed to the higher signal ion intensity 
 of the online mudpit method that maintained a constant s / n ratio between 
 tested groups . impact of peptide loss and ion suppression of background 
 noise 
 on mudpit platforms .
( b ) real - time monitoring of peptide elution 
 using 224 nm laser - induced native fluorescence ( linf ) detector .
( c ) 
 modified bicinchoninic ( bca ) method for peptide fractionated with 
 different mudpit platforms just before mass spectrometry .
( d ) signal - to - noise 
 ( s / n ) ratio represents the intensity of the signal ( peak height ) to 
 the intensity of the background noise ( in root - mean - square  rms  ) 
 for mudpit platforms .
red is online mudpit , blue is offline mudpit , 
 and black is offline - as mudpit .
it has long been known that peptides and 
 proteins can be readily 
 lost to surface adsorption to the materials they come in contact with . 

this issue was particularly troublesome when trying to purify proteins 
 or peptides to homogeneity for analysis because these methods often 
 required much sample manipulation .
ultimately the sample losses associated 
 with gel purification and in - gel - digestion - limited detection of proteins 
 made these methods less attractive .
one advantage of shotgun proteomics 
 is the preparation of proteins en masse for analysis by the mass spectrometer . 
 by preparing samples as a complex mixture
, the more abundant proteins 
 can act as carriers of the less abundant proteins .
after digestion , 
 the complex mixture of peptides needs to be separated by hplc for 
 introduction into the mass spectrometer .
several strategies have evolved 
 to fractionate peptide mixtures prior to entry into the mass spectrometer .
we demonstrated that the use of automated online mudpit results 
 in more comprehensive peptide separation and substantially more protein 
 and peptide identification in a label - free quantification experiment , 
 although results were less striking for tmt quantification , where 
 the nature of the experimental design may have complicated the comparison . 

differences attributed to sample loading are alleviated by normalization 
 correction in both experiments , together with comparable ms / ms spectra 
 ( similar s / n ratio ) , so it is likely that stochastic peptide loss 
 due to adsorption could be affecting offline sample collection ( such 
 as tubes and vials ) .
certainly , this conclusion does not discourage 
 using offline platforms because each format has its inherent advantages 
 and disadvantages ( i.e. , flexibility of offline fraction collection 
 and reduced labor time in online separation ) .
however , because adsorption 
 is a concentration - dependent surface phenomenon , one should critically 
 consider the potential sample loss due to surface adsorption when 
 considering offline fractionation platforms , especially when processing 
 minute quantities of valuable samples .
 large - scale 
 proteomics often employs two orthogonal separation 
 methods to fractionate complex peptide mixtures . 
 fractionation can 
 involve ion exchange separation coupled to reversed - phase separation 
 or , more recently , two reversed - phase separations performed at different 
 ph values . when multidimensional separations are combined with tandem 
 mass spectrometry for protein identification , the strategy is often 
 referred to as multidimensional protein identification technology 
 ( mudpit ) . 
 mudpit has been used in either an automated ( online ) or 
 manual ( offline ) format . in this study 
 , we evaluated the performance 
 of different mudpit strategies by both label - free and tandem mass 
 tag ( tmt ) isobaric tagging . 
 our findings revealed that online mudpit 
 provided more peptide / protein identifications and higher sequence 
 coverage than offline platforms . when employing an off - line fractionation 
 method with direct loading of samples onto the column from an eppendorf 
 tube via a high - pressure device , a 5.3% loss in protein identifications 
 is observed . when off - line fractionated samples are loaded via an 
 autosampler , 
 a 44.5% loss in protein identifications is observed compared 
 with direct loading of samples onto a triphasic capillary column . 
 
 moreover , peptide recovery was significantly lower after offline fractionation 
 than in online fractionation . 
 signal - to - noise ( s / n ) ratio , however , 
 was not significantly altered between experimental groups . 
 it is likely 
 that offline sample collection results in stochastic peptide loss 
 due to noncovalent adsorption to solid surfaces . 
 therefore , the use 
 of the offline approaches should be considered carefully when processing 
 minute quantities of valuable samples . 
Introduction
Materials and Methods
Results and Discussion
Conclusions