trypsin ( sequencing grade ) was 
 purchased from promega ( madison , wi ) .
isotope - labeled peptides were 
 obtained from new england peptide ( gardner , ma ) with either u  c6 , u 
n2-lysine 
 ( + 8 da ) or u  c6 , u  n4-arginine ( + 10 da ) at the peptide c terminus . chemical 
 purity was ranged from 95 to 99% and isotopic purity was greater than 
 99% .
timp1 , thbs2 , comp , msln , eng , mmp9 antibodies 
 and elisa kits were purchased from r&d systems ( minneapolis , mn ) . 

timp1 , eng and mmp9 recombinant proteins were purchase from sino biological 
 ( beijing , china ) .
identities of all recombinant proteins were confirmed 
 by sds - page ( figure s1a [ supporting information ( si ) ] ) and tandem mass spectrometry ( figure s2s7 [ si ] ) analysis of tryptic digests .
all other chemical 
 reagents were purchased from commercial sources and were used without 
 further purification .
human plasma samples 
 were collected during surgery for either colon carcinoma or for inguinal 
 hernia repair in accordance with the ayers institute protocol at vanderbilt 
 university medical center ( irb#110877 ) .
samples from colon cancer 
 patients undergoing surgery at vanderbilt university between august 
 2011 and june 2012 with a successful collection were used in the study . 

control samples were chosen from a larger group of inguinal hernia 
 repair patients who underwent surgery during the same time period . 

peripheral whole blood samples were collected preoperatively in edta 
 lavender top vacutainer tubes ( bd vacutainer , catalog number 366643 ) 
 and gently mixed by inverting the tube 810 times .
plasma was 
 separated by centrifugation at 1500 g for 10 min at 
 4 c .
aliquots ( 0.2 ml ) were taken and stored at 80 c 
 until needed .
antibodies were immobilized 
 on aldehyde beads ( thermo scientific , catalog number 26148 ) according 
 to the manufacturer s protocol with minor modifications .
briefly , 
 antibodies were dissolved in pbs buffer ( 0.01 m sodium phosphate , 
 0.15 sodium chloride , ph 7.2 ) and incubated with coupling resin and 
 75 m sodium cyanoborohydride at room temperature on a rotator . 

an aliquot was collected before and after binding for determination 
 of binding efficiency by protein bicinchoninic acid assay .
after immobilization , 
 the active aldehyde sites on the resin were blocked with 1 m tris 
 buffer and 75 m sodium cyanoborohydride followed by several 
 washes with pbs to remove any nonbound antibody . after determining 
 the binding efficiency ,
the immobilized resins for all antibodies 
 were either combined or directly aliquoted such that 1 g 
 of each immobilized antibody was used for each immunoprecipitation .
plasma 
 ( 50 l ) was diluted 5-fold with ripa buffer containing a protease 
 inhibitor cocktail ( roche , catalog number 11873580001 ) . diluted plasma 
 was incubated with the immobilized antibody resin overnight at 4 c 
 with gentle shaking .
the resin was washed three times with 0.5 ml 
 ripa buffer , and the bound proteins were eluted into 15 l of 
 2x nupage lithium dodecyl sulfate loading buffer ( invitrogen , carsbad , 
 ca ) containing 50 mm dtt by incubation at 95 c for 5 min . the 
 eluted proteins then were loaded and separated by sds - page on a nupage 
 novex 10% bis tris mini gel ( invitrogen np0301box ) .
a protein molecular 
 weight standard ( precision plus protein kaleidoscope standard , bio - rad , 
 hercules , ca ) was loaded in one lane on each gel and used for estimation 
 of relative mass determination of captured proteins .
after electrophoresis 
 at a constant 180 v for 20 min , gels were washed three times with 
 deionized water , stained with simplyblue safestain ( invitrogen ) for 
 1 h , and destained with deionized water at 4 c overnight . from 
 each gel lane , fractions were taken to enable targeted analysis of 
 the target proteins . for timp1 , a molecular weight fraction of 2537 
 kda was collected . for analysis of the remaining five proteins , a 
 molecular weight range of 75200 kda thbs2 , comp and mmp9 and 
 another of 3775 kda for eng and msln were excised from the 
 gel , cut into 1 mm cubes , and placed in 100 l of 100 mm ammonium 
 bicarbonate .
samples were reduced with 5 l of 100 mm dtt for 
 15 min at 50 c and alkylated with 15 l of 100 mm iodoacetamide 
 for 30 min at room temperature in the dark .
excess dye was removed 
 from gel slices with two exchanges of 100 l 50% acetonitrile/50 
 mm ammonium bicarbonate and subsequently dehydrated with 100% acetonitrile . 

the residue 
 was resuspended in 0.01 g/l ms grade trypsin ( promega , 
 madison , wi ) in 25 mm ammonium bicarbonate containing a standard mixture 
 of heavy isotope - labeled peptides for the analytes ( 20 fmol / peptide ) 
 and incubated at 37 c overnight .
peptides were extracted with 
 60% acetonitrile containing 1% formic acid , and then each fraction 
 was evaporated under vacuum .
samples were redissolved for mrm analysis 
 in 30 l of 5% acetonitrile containing 0.1% formic acid .
mrm analyses were performed on a tsq vantage 
 triple quadrupole mass spectrometer ( thermofisher scientific , san 
 jose , ca ) equipped with an eksigent ultra nanolc solvent delivery 
 system , microautosampler and a nanospray source .
sample peptides ( 3 
 l injection volume ) were loaded onto a 75 m  11 
 cm picofrit column ( pf360 - 75 - 10-n-5 , new objective , inc . ,
woburn , 
 ma ) packed with 3 m , reprosil - pur c18-aq ( dr . maisch , ammerbuch - entringen , 
 germany ) .
liquid chromatography was carried out at a flow rate of 
 300 nl / min with a mobile phase consisting of 0.1% formic acid in either 
 hplc grade water ( solvent a ) or 90% acetonitrile ( solvent b ) .
an elution 
 gradient was programmed from 97% a for 1 min , then increased to 7% 
 b over 4 min , 25% b over 15 min , 40% b over 7 min , 90% b by 40 min , 
 and then held at that composition for 10 min before returning to 97% 
 solvent a over 1 min .
mobile phase composition then was held at this 
 initial condition for 29 min prior to the next analysis .
instrument 
 parameters included q2 gas 1.5 mtorr , scan width 0.005 th , scan time 
 10 ms , and both q1 and q3 resolution fwhm 0.7 .
proteotypic peptides 
 and mrm transitions were selected with the skyline software utility and were further optimized by analyses of tryptic 
 digests of recombinant proteins on the tsq vantage triple quadrupole 
 mass spectrometer .
we digested the recombinant proteins 
 and determined the resultant peptides by monitoring all possible tryptic 
 peptides between 8 and 22 amino acids in lc 
ms experiments 
 using the product scan mode . where recombinant protein was not available , 
 selection of peptides and transitions were selected on the basis of 
 observations from previous discovery experiments , observed peptides 
 in open proteomics databases , or computational predicted peptides 
 through algorithms such as enhanced signature peptide ( esp ) predictor .
peptides containing methionine residues as well 
 as those containing post - translational modification sites such as 
 glycosylation listed in the uniprot database ( to minimize interference 
 on digestion ) were excluded .
all peptides were run through blastp 
 ( uniprot ) to ensure their uniqueness to the protein of interest .
peptides 
 and transitions selected for each protein are shown in table s1 ( si ) .
mrm data acquired on the tsq vantage 
 triple quadrupole mass spectrometer were analyzed with skyline software .
peak integrations were manually reviewed and 
 transitions from peptides measured were confirmed by the same retention 
 times and transition patterns of the light peptides and synthetic 
 heavy , stable isotope - labeled peptides .
peak areas for the four most 
 intense mrm transitions were integrated and summed to generate a peptide 
 peak area , which was divided by the peptide peak area for the internal 
 standard heavy peptide . for each protein , the unique peptide that 
 generated the highest summed peak area signal was used to quantify 
 the protein of interest .
six biomarker candidate proteins ( timp1 , eng , msln , thbs2 , mmp9 and 
 comp ) were selected on the basis of literature reports , which suggest 
 that they are overexpressed in colon cancers or because differential 
 expression of these proteins in blood was associated with cancer .
we also chose these candidates because well - characterized , commercially 
 available elisas were available for each .
moreover , the same capture 
 antibodies used in the elisas were available to us for evaluation 
 in ip - mrm assays . to analyze the biomarker candidates in plasma ,
we 
 performed either single - protein or multiplexed immunoprecipitations 
 and then resolved the target proteins into molecular weight fractions 
 by sds - page ( figure s1b [ si ] ) .
the gel 
 bands were digested and the digests were spiked with isotope - labeled 
 standards and analyzed by mrm .
the mrm signals from each peptide were 
 shown to be consistent with their corresponding isotope labeled peptides 
 in both retention time and transient patterns ( figure s8 [ si ] ) .
recombinant timp1 protein was spiked into a  mock 
 plasma  matrix consisting 60 mg / ml bsa in pbs to mimic the 
 human plasma environment and provide a defined analyte concentration 
 for estimation of recovery . a 2-fold dilution series ranging from 
 640 to 10
ng / ml was constructed and a plot showing the theoretical 
 timp1 concentration versus the calculated protein concentration is 
 shown in figure 1a ( the theoretical timp1 concentration 
 versus the ratio l / h is also shown in figure s9a ( si ) .
the calculated protein concentration was calculated from 
 the measured peak area ratio of the light to heavy peptides .
the slope 
 ( 0.329 ) indicated timp1 recovery after all process steps including 
 immunoaffinity capture , trypsin digestion and peptide extraction . 

the limit of detection ( lod ) was 2.5 ng / ml ( table 1 ) .
response curves for ip - mrm analyses of recombinant timp1 , comp , 
 mmp9 , thbs2 , msln and eng proteins .
proteins were spiked at 10640 
 ng / ml in a background matrix of 60 mg / ml bsa in dpbs and analyzed 
 by ip - mrm as described in experimental procedures .
values are mean  sd . the elisa lower limit of quantitation 
 ( lloq ) was based on the lowest concentration for the manufacturer s 
 specified calibration curve .
next , ip - mrm was used to measure timp1 in 12 50 l plasma 
 aliquots from patients with colon cancer and in 12 samples from noncancer 
 controls .
the same samples were also analyzed with a commercial elisa 
 kit with the same capture antibody ( figure s10a [ si ] ) .
the mean values in ip - mrm analyses in plasma samples 
 from cancer patients and controls were 212 ng / ml and 141 ng / ml , respectively , 
 which was a significant difference ( unpaired t test , p = 0.0028 ) ( table 1 ) . in elisa analyses , 

the mean timp1 levels in plasma samples from cancer patients and controls 
 were 120 ng / ml and 97 ng / ml , which were not significantly different 
 ( unpaired t test , p = 0.06 ) .
a 
 key advantage of mrm - based methods is the capacity for multiplexed 
 analyses , which can increase analysis throughput and minimize sample 
 consumption .
we performed a multiplexed ip - mrm analysis of the remaining 
 proteins ( eng , msln , thbs2 , mmp9 and comp ) , which were spiked into 
 a  mock plasma  matrix consisting of 60 mg / ml bsa in 
 pbs to produce concentrations ranging from 640 to 10 ng / ml .
antibodies 
 for the five proteins ( one antibody for each protein ) were immobilized 
 on the resin , mixed and aliquoted .
the mixed antibody resin ( 1 g / antibody / analyte ) 
 was incubated with solutions of bsa matrix containing the spiked target 
 proteins .
plots of calculated protein concentration determined by the peak area 
 ratio of the light peptide to heavy peptide ( l / h ) showed a linear 
 increase in measured protein across the concentration range ( figure 1b  f ) ( the theoretical protein concentration 
 versus the ratio
l / h is also shown in figure s9b  f [ si ] ) .
recoveries were determined for each 
 of the five proteins based on the slope of the response curve and 
 ranged from 13.5% ( thbs2 ) to 55% ( msln ) .
. this could reflect 
 less efficient capture or digestion for these proteins , or some combination 
 of both effects .
lod values were determined by comparing the variance 
 of the blank samples ( with no analyte spiked in ) to the variance of 
 the lowest level spiked sample ( analyte at 10 ng / ml ) .
the lod values were between 2 and 10 ng / ml ( table 1 ) .
on the basis of triplicate measurements at the 
 lowest spiked concentration ( 10 ng / ml ) for each protein , cv values 
 were all below 20% ( table 1 ) .
all five proteins were analyzed individually by elisa in plasma 
 samples from 12 cancer patients and from 12 controls ( 11 cancer plasma 
 samples were analyzed for timp1 and mmp9 ) . the mean concentrations 
 of these five proteins determined by elisa in plasma samples from 
 normal and cancer patients ranged from 3.9 ng / ml ( eng ) to 208 ng / ml 
 ( comp ) ( figure s10b  f [ si ] ) , table 1 ) . for ip - mrm analyses ,
the mixed antibody resin 
 ( 1 g antibody / analyte ) was used to simultaneously capture all 
 five protein targets .
three aliquots of each colon cancer and normal 
 plasma sample were analyzed by ip - mrm .
the mean concentrations of 
 these five proteins determined by ip - mrm ranged from 8.3 ng / ml ( eng ) 
 to 221 ng / ml ( comp ) ( figure s10b  f [ si ] ) , table 1 ) .
comparison of measured values 
 between normal and cancer plasma samples with either elisa or ip - mrm 
 yielded only two significant differences ( p < 
 0.05 , unpaired student t test ) for timp1 and thbs2 , 
 but only when measured by ip - mrm ( table 1 )
. we asked how the ip - mrm and elisa methods compared 
 for measurement of the six biomarker candidate proteins across individual 
 samples .
figure 2 shows paired comparisons 
 of measurements with the two analysis methods for each sample .
the 
 data indicate that the concordance of measurements depends on both 
 the analyte and the method .
analysis of the average measured plasma 
 concentrations for all analytes and all samples indicated that the 
 ip - mrm measurements were higher by 8.6 ng / ml at ( p = 0.005 , paired t test ; 95% ci : 2.714.5 ) , 
 across all markers and that the two measurements were highly correlated 
 ( r = 0.93 ) .
comparison of the average difference 
 in plasma concentrations measured by ip - mrm versus elisa for individual 
 candidates indicated that differences were significant ( p < 0.05 , paired t test ) for timp1 ( 67.1 ng / ml ) , 
 thbs2 ( 13.4 ng / ml ) , msln ( 11.7 ng / ml ) and eng ( 5.2 
 ng / ml ) , but not for comp ( 6.4 ng / ml ) , mmp9 ( 13.5 ng / ml ) .
we 
 also examined correlation of ip - mrm and elisa for the individual analytes 
 ( figure 3 ) .
correlation coefficients for the 
 two methods were high for mmp9 , comp and timp1 and moderate for thbs2 
 and msln .
however there was no apparent correlation between the methods 
 for eng , as elisa measurements all were approximately 5 ng / ml , whereas 
 ip - mrm measurements varied from 5 to 20 ng / ml .
quantitation of six biomarker candidate proteins in plasma samples 
 from colon cancer patients and noncancer controls by ip - mrm and elisa . 

plasma levels of timp1 , comp , mmp9 , thbs2 , msln and eng were measured 
 in controls ( circles ) and colon cancer patients ( squares ) .
results 
 are from the average of triplicate ip - mrm measurements and duplicate 
 elisa measurements .
correlation of protein expression levels measured by elisa and 
 ip - mrm in plasma samples .
mean concentrations from triplicate analyses 
 of timp1 , comp , mmp9 , thbs2 , msln and eng in plasma samples from 24 
 subjects ( 23 for timp1 and mmp9 ) by elisa and ip - mrm are plotted . 

the targeted analysis of protein biomarker candidates in plasma 
 or serum requires highly sensitive and specific assays .
although elisa 
 provides the gold standard platform for such analyses , the selection 
 of high - quality elisas is limited .
configuration of a new elisa is 
 often costly and time - consuming and may fail for lack of high - quality 
 reagents . the rationale for hybrid immuno 
ms assays was first 
 proposed by anderson and colleagues and 
 has been developed through the peptide capture ( siscapa ) approach .
the key advantages of an immuno  ms approach 
 are the ability to systematically configure mrm assays based on proteotypic 
 peptides and the requirement for only one antibody for target capture . 

protein - capture - based immuno  ms analysis has been reported , but has not been as thoroughly explored .
a potential advantage of 
 protein capture is that the antibodies might be employed to transition 
 the ip - mrm assay to an elisa platform . with that consideration in 
 mind
, we asked how ip - mrm and elisa would compare when the same capture 
 antibodies are used .
our results indicate that the two methods offer equivalent performance 
 with a few exceptions .
a global comparison of the ip - mrm and elisa 
 measurements in our study indicated a slight systematic bias in favor 
 of higher measurement values for ip - mrm .
however , this difference 
 was not predictive for individual analytes , as absolute measurement 
 differences between the methods varied in either direction by approximately 
 510 ng / ml .
measurement variation ( cv at lod ) for ip - mrm ranged 
 from 2.3 to 19% , which was similar to variation for elisas of the 
 same samples ( table 1 ) .
ip - mrm and elisa measurements 
 were well - correlated , with coefficients for all of the analytes except 
 eng = .017 .
poor correlation of ip - mrm and elisa for eng appears 
 to be related to limited range of the elisa measurements , which were 
 near the lod of the assay at the dilution used ( figure s10f [ si ] ) .
because the same capture antibody was used 
 for both assays , it appears unlikely that differential capture would 
 explain the difference in eng measurement by the two methods .
the 
 difference may reflect selective recognition of a subset of eng proteins 
 in plasma samples by the detection antibody , perhaps due to unanticipated 
 modifications . such modifications may not have affected peptide - based 
 quantitation in ip - mrm assays .
we used plasma samples from individuals with colon cancers and 
 from noncancer controls in our study , and the biomarker candidate 
 proteins were selected on the basis of existing literature .
however , 
 these experiments were designed only to compare the performance of 
 the assay platforms , not to validate biomarker candidates for colon 
 cancer detection .
although we can not draw any conclusions about the 
 utility of the biomarker candidates , the data illustrate the importance 
 of reliable , precise assays for biomarker validation studies .
we observed 
 a high degree of overlap in measurement distributions for all of the 
 candidates in control and cancer plasma samples .
this is expected 
 for most biomarker candidates that , despite being overexpressed in 
 tumors , also may be derived from other tissues .
assays capable of 
 validating cancer biomarkers will be expected to precisely measure 
 small concentration differences for proteins present at ng / ml levels 
 or lower .
our study affirmed a key advantage of ip - mrm , which is the ability 
 of the assay platform to perform multiplexed protein capture , as reported 
 by nico et al .
this advantage is particularly 
 important when amounts of plasma or serum samples are limited . in 
 our study , all proteins could be captured and quantified from a single 
 aliquot of 50 l plasma , while input quantities for single elisa 
 measurements were between 2 and 66 l . the capture antibody 
 for ip - mrm approach should have a relatively high affinity for the 
 targeted protein .
our experiments represent a  best case  
 example , because the capture antibodies were of sufficiently high 
 quality to support robust and sensitive elisa kits . we have done other 
 ip - mrm experiments with antibodies found to be unsatisfactory for 
 elisa , and the same antibodies also failed to provide significant 
 protein enrichment for ip - mrm measurements ( unpublished observations ) . 

ip - mrm might be used to analyze different isoforms or post - translational 
 modifications of proteins if they are bound with similar affinity 
 by the capture antibody .
another potentially advantageous feature of ip - mrm is the detection 
 of proteins via multiple peptides .
while this offers flexibility in 
 assay development and confirmation of measurements made on single 
 peptides , previous work demonstrates that one peptide usually provides 
 the greatest measurement sensitivity .
for this reason , 
 we chose not to explore comparison mrm measurements with multiple 
 peptides and instead focused on analysis of peptides that generated 
 the strongest signals .
in addition , higher throughput ip - mrm assays 
 can utilize magnetic beads for antibody capture in a 96-well - plate 
 format .
ip - mrm directed at intact proteins provides an effective approach 
 to systematically configure targeted protein measurements .
the protein - targeted 
 capture compares favorably to both peptide - targeted ip - mrm and elisa . 
 for novel targets ,
the choice of methods depends primarily on the 
 availability of antibodies and their performance .
protein - based ip - mrm 
 assays offer a faster , less costly approach to targeted protein measurement 
 than elisas and could dramatically expand the scope of targeted protein 
 quantitation in biology and medicine .
 quantitative analysis of protein biomarkers in plasma is typically 
 done by elisa , but this method is limited by the availability of high - quality 
 antibodies . 
 an alternative approach is protein immunoprecipitation 
 combined with multiple reaction monitoring mass spectrometry ( ip - mrm ) . 
 
 we compared ip - mrm to elisa for the analysis of six colon cancer biomarker 
 candidates ( metalloproteinase inhibitor 1 ( timp1 ) , cartilage oligomeric 
 matrix protein ( comp ) , thrombospondin-2 ( thbs2 ) , endoglin ( eng ) , mesothelin 
 ( msln ) and matrix metalloproteinase-9 ( mmp9 ) ) in plasma from colon 
 cancer patients and noncancer controls . 
 proteins were analyzed by 
 multiplex immunoprecipitation from plasma with the elisa capture antibodies , 
 further purified by sds - page , digested and analyzed by stable isotope 
 dilution mrm . 
 ip - mrm provided linear responses ( r = 0.9780.995 ) between 10 and 640 ng / ml for the target proteins 
 spiked into a  mock plasma  matrix consisting of 60 
 mg / ml bovine serum albumin . 
 measurement variation ( coefficient of 
 variation at the limit of detection ) for ip - mrm assays ranged from 
 2.3 to 19% , which was similar to variation for elisas of the same 
 samples . 
 ip - mrm and elisa measurements for all target proteins except 
 eng were highly correlated ( r = 0.670.97 ) . 
 
 ip - mrm with high - quality capture antibodies thus provides an effective 
 alternative method to elisa for protein quantitation in biological 
 fluids . 
Experimental Procedures
Results
Discussion