a growing body of literature has provided 
 robust evidence that 
 formalin fixed , paraffin - embedded ( ffpe ) tissue can be successfully 
 analyzed using mass spectrometry - based proteomic methods , enabling 
 the use of these archival specimens for biomarker discovery through 
 retrospective analysis .
although qualitative protein identifications can be obtained from 
 fixed tissue , fixation leads to covalent chemical modification and 
 cross - linking of proteins , dna and rna , which may be expected to affect 
 the quantitative reliability of ffpe tissue analyses by targeted proteomic 
 methods , such as multiple reaction monitoring ( mrm ) .
recently , research 
 efforts have begun to demonstrate the technical feasibility of targeted 
 quantitative proteomic analysis in ffpe tissue .
huang et al . 
 described the shotgun proteomic analysis of laser capture microdissected 
 ffpe primary and metastatic melanomas and identified 120 proteins 
 as potential markers of metastasis .
label - free 
 quantitation was performed with extracted ms1 signal from an ion trap 
 ms instrument .
analyzed calcyclin peptides by 
 selected reaction monitoring ( one transition per peptide ) on a triple 
 quadrupole instrument with sequence analogue peptides as reference 
 standards .
similarly , nishimura et al . 
 quantified two potential prognostic markers for lung adenocarcinoma 
 in ffpe tissue using an endogenous -actin peptide as an internal 
 standard .
although these reports have 
 demonstrated the application of targeted protein analyses of ffpe 
 tissue , the critical issue of how formalin fixation affects the precision 
 of quantitative analyses in ffpe tissues remains unexplored . because 
 formaldehyde chemistry significantly affects lysine c - terminal peptides , 
 it is not clear whether quantitative strategies must be adjusted to 
 avoid these . to assess the impact of fixation on the reproducibility 
 of peptide 
 quantitation
, we compared the mrm signals for tryptic peptides derived 
 from paired fixed and frozen clear cell renal cell carcinoma ( rcc ) 
 tissue from the same tumor .
our analyses describe the precision of 
 measurements of a large set of tryptic peptides across a broad concentration 
 range in both ffpe and frozen specimens .
we further evaluated the 
 precision of mrm - based quantitation through the analysis of peptides 
 from the her2 ( erbb2 ) receptor in ffpe and frozen tissues from a mouse 
 xenograft model of her2 overexpressing breast cancer .
finally , we 
 used mrm analyses to quantify her2 protein in ffpe specimens of immunohistochemically 
 confirmed her2-positive and her2-negative human breast cancers .
sub - x xylene substitute was 
 obtained from surgipath ( richmond , il ) .
iodoacetamide ( iam ) was from 
 sigma ( st . louis , mo ) , tris - carboxyethylphosphine 
 ( tcep ) was from pierce ( rockford , il ) , sequencing grade trypsin was 
 from promega ( madison , wi ) , trifluoroethanol and dithiothreitol ( dtt ) 
 were from acros ( geel , belgium ) .
trifluoroacetic acid , ammonium bicarbonate , 
 and urea were purchased from thermofisher scientific ( waltham , ma ) .
the deidentified human tissue samples 
 and experimental protocol were subject to institutional review board 
 exempt approval ( institutional review board protocols 080856 and 110453 ) . 
 fixed and frozen rcc tissues were obtained from the cooperative human 
 tissue network - western division ( vanderbilt university , nashville , 
 tn ) .
ffpe human breast cancer tissue blocks were obtained through 
 the vanderbilt - ingram cancer center breast cancer spore tissue core . 
 fixed and
frozen mouse xenografts of bt474 ( her2 + ) and sum159 ( her2 
 negative ) cell lines were kindly provided by the laboratory of carlos 
 l. arteaga .
paraffin was removed with three washes of 1 ml of 
 sub - x and tissue was rehydrated with 2  1 ml washes each of 
 100% , 85% , and 70% ethanol .
deparaffinized , rehydrated tissue slices 
 were resuspended in 100 l of ammonium bicarbonate ( 100 mm , 
 ph 8.0 ) and were heated at 80 c for 2 h. tryptic digestion was 
 done by an adaptation of the method of wang et al .
trifluoroethanol ( tfe ) ( 100 l ) was added and the 
 samples were sonicated for 20 s followed by 30 s incubation on ice . 

the resulting homogenate was heated 
 for 1 h at 60 c followed by a second series of sonication steps , 
 as described above .
the homogenate was reduced with tris-(carboxyethyl)phosphine ( 10 mm ) and dithiothreitol ( 25 mm ) at 60 
 c for 30 min , followed by alkylation with iodoacetamide ( 50 
 mm ) in the dark at ambient temperature for 20 min .
the reduced and 
 alkylated protein mixture was diluted to 1 ml with ammonium bicarbonate 
 ( 50 mm , ph 8.0 ) followed by addition of trypsin at 1:50 ( w / w ) .
the 
 digest was incubated overnight at 37 c , followed by freezing 
 at 80 c and lyophilization .
samples were resuspended 
 in 1 ml of water , desalted over 1 cm ( 100 mg ) sep - pak 
 vac c-18 cartridges ( waters corp . ,
milford , ma ) , and evaporated to 
 dryness in vacuo with a speed - vac sample concentrator ( thermofisher , 
 waltham , ma ) .
isoelectric focusing 
 ( ief ) of tryptic peptides was performed by a modification of the method 
 described previously .
rcc tryptic 
 peptides ( 200 g ) were resuspended in 500 l of 6 m urea 
 and loaded in an ipgphor rehydration tray .
immobiline immobilized 
 ph gradient strips ( 24 cm , ph 3.54.5 ) were placed over the 
 samples and allowed to rehydrate overnight at ambient temperature . 

the loaded strips were focused at 21 c on an ettan ipgphor-3 
 ief system ( ge healthcare , piscataway , nj ) using the following program : 
 step at 300 v for 900 vh ; gradient to 1000 v for 3900 vh ; gradient 
 to 8000 v for 13500 vh ; step to 8000 v for 93  700 vh .
the strips 
 were then cut into 20 ( 1.2 cm ) pieces and placed in separate wells 
 of a 96-well elisa plate .
peptides were eluted from the strips as 
 follows : 200 l of 0.1% formic acid ( fa ) for 15 min ; 200 l 
 of 50% acetonitrile ( acn)/0.1% fa for 15 min ; 200 l of 100% 
 acn/0.1% fa for 15 min .
solutions of extracted peptides were evaporated 
 in vacuo , resuspended in 1 ml 0.1% trifluoroacetic acid , and desalted 
 over a 96-well c18 oasis hlb plate 30 m ( 10 mg ) ( waters corp . , 

peptide solutions were evaporated in vacuo , resuspended 
 in 100 l of 0.1% fa , and placed in sample vials for liquid 
 chromatography  tandem mass spectrometry ( lc  ms / ms ) analysis .
ms / ms 
 analyses were performed on an ltq - xl mass spectrometer ( thermo electron , 
 san jose , ca ) equipped with an eksigent 1d nanolc and microautosampler 
 ( dublin , ca ) .
peptides were loaded on a 100 m  5 cm fused 
 silica capillary guard column ( polymicro technologies , llc . ,
phoenix , 
 az ) packed with 5 m , 300  jupiter c18 ( phenomenex , torrance , 
 ca ) and resolved on a 100 m  11 cm fused silica capillary 
 column ( polymicro technologies , llc . , phoenix , az ) packed with 5 m , 
 300  jupiter c18 ( phenomenex , torrance , ca ) .
liquid chromatography 
 was carried out at ambient temperature at a flow rate of 0.6 l 
 min using a gradient mixture of 0.1% ( v / v ) formic 
 acid in water ( solvent a ) and 0.1% ( v / v ) formic acid in acn ( solvent 
 b ) .
peptides eluting from the capillary tip were introduced into the 
 ltq source in microelectrospray mode with a capillary voltage of approximately 
 2 kv .
a full scan was obtained for eluting peptides in the range of 
 4002000 amu followed by six data - dependent ms / ms scans .
ms / ms 
 spectra were recorded using dynamic exclusion of previously analyzed 
 precursors for 60 s with a repeat of 1 and a repeat duration of 1 . 

ms / ms spectra were generated by collision induced dissociation of 
 the peptide ions at a normalized collision energy of 35% to generate 
 a series of b- and y - ions as major fragments .
mrm analyses were performed on a tsq vantage 
 triple quadurpole mass spectrometer ( thermo electron , san jose , ca ) 
 equipped with an eksigent ultra nanolc and microautosampler ( dublin , 
 ca ) .
ms / ms 
 analyses , except that the flow rate was reduced to 0.4 l min .
analyses were performed using the labeled referenced 
 peptide ( lrp ) method , where peak areas 
 for target peptides were normalized against the isotope labeled -actin 
 peptide standard u 
c , n - arg - gysftttaer , 
 which was added at a concentration of 25 nm .
four transitions were 
 monitored for each peptide and a maximum of 40 peptides were monitored 
 per method . extracted ion chromatogram peak areas were measured as 
 the sum of the peak areas for the four monitored transitions .
transitions 
 were selected from the most intense y - ions observed in a spectral 
 library derived from shotgun data sets .
the scansifter algorithm read tandem 
 mass spectra stored as centroided peak lists from thermo raw files 
 and transcoded them to mzml files . if 
 90% of the intensity of a tandem mass spectrum appeared at a lower m / z than that of the precursor ion , a single 
 precursor charge was assumed ; otherwise , the spectrum was processed 
 under both double and triple precursor charge assumptions .
tandem 
 mass spectra were assigned to peptides from the ipi human database 
 version 3.37 ( may 5 , 2008 ; 69  238 sequences ) by the myrimatch 
 algorithm , version 1.6.75 .
the sequence 
 database was doubled to contain each sequence in both normal and reversed 
 orientations , enabling false discovery rate estimation .
myrimatch 
 was configured to expect all cysteines to bear carboxamidomethyl modifications 
 and to allow for the possibility of oxidation on methionines .
candidate 
 peptides were required to feature trypsin cleavages or protein termini 
 at one end ( semitryptic search ) ; any number of missed cleavages was 
 permitted .
a precursor error of 1.25 m / z was allowed , but fragment ions were required to match within 0.5 m / z .
the idpicker algorithm v2.6.165.0 filtered the identifications for each lc  ms / ms 
 run to include the largest set for which a 5% identification false 
 discovery rate could be maintained , as described by qian et al .
, and applied parsimonious protein assembly , reporting 
 the smallest list of proteins which could account for the identified 
 peptides .
proteins were required to have at least two different peptide sequences 
 observed within an ief sample set .
database protein entries that could 
 not be distinguished based on the observed peptides were combined 
 into  protein groups  representing the most parsimonious 
 assignment of the spectral count data .
false discovery rates ( fdr ) 
 rates were computed by the formula : the algorithm reported the number of spectra 
 and number of distinct sequences observed for each protein and protein 
 group in each sample set .
shotgun proteome 
 analysis of fixed and frozen rcc tissue indicated a qualitative concordance 
 in protein groups identified in both ffpe and frozen tissue ( figure 1a ) , as we reported previously for comparison of 
 frozen and ffpe tissues .
( spectral count 
 data for the identified proteins is presented in supporting information table s1 ; a full summary of the data 
 set is provided in the accompanying idpicker report .
of 2165 total 
 protein groups identified , 91% were found in both sample types .
however , 
 the resulting peptide identifications obtained from tryptic digests 
 of fixed tissue were biased against lysine c - terminal peptides ( figure 1b ) .
whereas in frozen tissue the ratio of lysine 
 c - terminal peptides to arginine c - terminal peptides was 1.11 , the 
 ratio in ffpe samples was reduced to 0.93 .
these observations are 
 in agreement with our previous analyses of frozen and ffpe colon adenocarcinoma 
 tissue and are consistent with the known 
 reactivity of aldehydes toward primary amines , which preferentially 
 consumes lysine residues and thus affects the yield of lysine c - terminal 
 peptides .
we thus asked whether the modification of lysine residues 
 would affect the quantitation of lysine c - terminal peptides in a tryptic 
 digest of ffpe tissue .
high 
 qualitative 
 concordance was observed for proteins identified in both ffpe and 
 frozen tissue digests .
peptides observed in ffpe tissue were biased 
 against lysine c - terminal peptides , indicated by a lower lysine to 
 arginine peptide ratio .
error bars represent standard deviation from 
 4 ief replicates . to assess measurement 
 precision in ffpe tissue , 114 peptides were selected as mrm targets 
 from a list of peptides identified from shotgun proteomic analysis 
 of ief - fractionated tryptic digests of ffpe and frozen rcc tissue 
 ( supporting information table s2 ) .
the 
 peptides selected spanned the entire range of peptide and protein 
 abundance , as determined by spectral count information in the shotgun 
 data set ( supporting information table s1 ) .
the 114 candidates each were between 7 and 25 amino acids in length ; 
 contained no methionine , cysteine , histidine , n - terminal glutamine 
 residues or missed tryptic cleavages ; were unique to a single protein 
 in the search database ; and were observed predominantly as doubly 
 charged precursors .
transitions were chosen from the 4 most intense 
 y - ions based on reference spectra from the shotgun data set .
the precision with which a peptide can be measured and the intensity 
 of the signal obtained are a function of the peptide concentration , 
 ionization and fragmentation characteristics , variability in yields 
 of sample processing steps ( e.g. , digestion ) , and the characteristics 
 of the instrument used to measure the analyte .
the measurement precision 
 of the instrument can be empirically assessed through the analysis 
 of an internal standard of known concentration and can provide a reference 
 for the measurement of effects on precision caused by the fixation 
 process .
thus , in addition to the 114 candidate peptides , an endogenous 
 peptide of sequence gysftttaer , corresponding to -actin , was 
 monitored , as well as an isotope labeled version of the gysftttaer 
 peptide spiked - in at a constant concentration of 25 nm to provide 
 for signal normalization for the targeted peptides and to monitor 
 measurement precision .
five serial 
 sections of ffpe and frozen rcc tissue were processed 
 in parallel and analyzed using 3 mrm acquisition methods , which monitored 
 40 peptides and 160 transitions each .
the methods analyzed groups 
 of peptide targets corresponding to proteins of different abundances , 
 based on spectral counts from the shotgun analyses .
median spectral 
 counts for the proteins covered in methods 1 , 2 , and 3 were 190 , 129 , 
 and 38 , respectively .
the proteins and peptides comprising the methods 
 are listed in supporting information table s3 .
lists of the transitions monitored for each peptide are provided 
 as skyline ( .sky ) files in the supporting information .
the coefficient of variance ( cv ) for the lrp - normalized peak area 
 over triplicate mrm runs was determined for each peptide . given 
 the possible effects of formaldehyde fixation chemistry on 
 lysine c - terminal tryptic peptides , the precision of mrm measurement 

was analyzed separately for lysine and arginine c - terminal peptides . 
 the median cvs for the 52 lysine c - terminal peptides were not significantly 
 different between analyses of fixed ( median cv = 0.195 ) or frozen 
 tissue ( median cv = 0.190 ) using the mann 
median cvs for analyses of the 58 arginine c - terminal 
 peptides also indicated no significant difference between fixed ( median 
 cv = 0.183 ) or frozen tissue ( median cv = 0.180 , figure 2 ) .
this result suggests that there is no discernible effect 
 of formaldehyde fixation chemistry that affects the reproducibility 
 of peptide mrm measurement .
in addition , there was no significant 
 difference in median cv between lysine c - terminal peptides ( median 
 cv = 0.195 ) and arginine c - terminal peptides ( median cv = 0.183 ) from 
 ffpe tissue , suggesting that mrm measurements of ffpe tryptic peptides 
 display similar technical variation , regardless of the c - terminal 
 amino acid .
cvs for mrm measurements of 52 lysine c - terminal peptides and 58 
 arginine c - terminal peptides in 5 serial sections of frozen and ffpe 
 rcc samples .
comparison 
 of median cvs for peptide measurements from ffpe and frozen tissues 
 indicated no significant effect of fixation on the precision of peptide 
 measurement ( mann  whitney u test ) .
the influence of fixation status on individual 
 peptides was further 
 assessed by considering the median cv obtained for analyses of each 
 peptide across the 5 serial slices ( supporting 
 information table s4 ) .
a commonly used method to establish 
 the limit of quantitation ( loq ) for an analyte is to determine the 
 average signal at the retention time of interest in a blank sample 
 plus 8 standard deviations of the average blank signal . for mrm analyses , signals from a blank run at 
 a given peptide m / z value and retention 
 time can result in very small values , which can yield theoretical 
 loqs far below the practical limitations of the analytical system . 

a more conservative loq estimate can be obtained empirically by determining 
 the concentration at which the cv of replicate measurements exceeds 
 25% .
of the 114 peptides analyzed , 10 were deemed 
 to be undetectable 
 due to lack of a consistent signal among replicates and samples . of 
 the remaining 104 peptides , 37 yielded median cvs greater than 25% 
 in the ffpe derived samples .
these peptides were thus designated as 
 being below the loq in these samples . in analyses of frozen tissue 
 digests , 27 peptides exceeded the 25% median cv threshold ( supporting information table s3 ) .
a greater number 
 of peptides below the loq in ffpe tissues may reflect reduced signal 
 intensity , most likely due to reduced tryptic peptide yield in digests 
 of ffpe samples . in total ,
46 peptides yielded median cvs in 
 excess of 25% in either 
 ffpe or frozen tissue ; 18 of these had median cvs greater than 25% 
 in both tissue types .
overall , 51% of the peptides targeted ( 58/114 ) 
 yielded signals estimated to be above the loq .
this success rate for 
 analyses in ffpe tissues is comparable to other empirical assessments 
 of proteotypic peptide suitability in semiquantitative mrm assay development 
 for biomarker verification .
as is evident 
 in supporting information table s3 , methods 
 2 and 3 produced increasing numbers of peptides exceeding the 25% 
 median cv threshold and this effect was somewhat greater for ffpe 
 tissue .
peptides exceeding the threshold were as follows : method 2 , 
 16 peptides in ffpe , 9 in frozen ; method 3 , 21 in ffpe , 18 in frozen . 

peptides targeted by these two methods were from proteins represented 
 with fewer spectral counts in the shotgun analysis ( supporting information table s2 ) , indicating lower abundance 
 in the rcc tissues .
the most likely explanation for the effect 
 on median cv in the 
 ffpe samples is the presence of protein cross - links , which result 
 in a lower yield of proteotypic tryptic peptides detectable by mrm 
 analysis , thus decreasing concentrations of the peptides below the 
 limit of quantitation .
the influence of fixation appears not to be 
 specific to a particular class of protein , or subcellular compartment , 
 but rather is distributed uniformly among proteins we analyzed . in 
 mrm analyses , effects on peptide yield will have the greatest impact 
 on quantification of peptides that are already near the limit of quantitation 
 in unfixed , frozen samples .
this interpretation is consistent with 
 our observation here ( figure 1 and supporting information table 1 ) and previously that the proteins represented in shotgun data 
 sets with fewer spectral counts yield fewer peptide and protein group 
 identifications in ffpe tissue than in frozen specimens in shotgun 
 proteomic analysis of ief - fractionated samples .
we also considered 
 the effect of fixation on the magnitude of the 
 peak areas observed during mrm analysis , a more direct measurement 
 of relative peptide abundance .
of the 114 peptides monitored , 70% 
 showed a significant difference in average normalized peak area between 
 fixed and frozen samples ( two tailed t test , p < 0.05 , supporting information 
 figure 1 ) . figure 3 plots the log2 ratio 
 of lrp - normalized peak areas for peptides from ffpe to frozen tissues . 

these data demonstrate that peptides from ffpe tissue generally yield 
 lower normalized average peak areas than peptides from frozen tissue , 
 given the predominance of log2 ( ffpe : frozen ) peptide intensity values 
 below zero in figure 3 .
this result further 
 illustrates the significant impact of fixation on the quantitation 
 of proteins in tissue .
peptides 
 are ordered on the x - axis in order of decreasing 
 log2 ratios ( y - axis values ) .
log2 ratios less than zero indicate higher 
 peak areas from peptides derived from frozen tissue . a potentially 
 useful application of mrm quantitative analyses in ffpe tissue is 
 the measurement of specific biomarker proteins of clinical interest . 

this is normally achieved by immunohistochemistry ( ihc ) , which can 
 be limited by assay variability , antibody sensitivity , and specificity . 

in addition , ihc methods are not quantitative and assessment of ihc 
 is subjective .
mrm analyses provide quantitative measurements with 
 the potential for improved specificity , thus overcoming key performance 
 limitations of ihc . to explore the possible clinical application of 
 mrm in ffpe tissue
amplification of her2 is 
 observed in 20% of breast cancer cases and patients harboring her2-overexpressing 
 tumors are candidates for her2-targeted therapies . to monitor 
 multiple peptides unique to her2
, the peptide exclusion criteria were 
 relaxed allowing for the inclusion of peptides containing cysteine , 
 methionine , and histidines .
peptides were still required to be fully 
 tryptic , between 7 and 25 amino acids in length , unique to her2 and 
 to be represented in a reference spectral library .
twenty - five peptides 
 met these criteria and were targeted by mrm in a tryptic digest of 
 the her2 overexpressing bt474 human breast cancer cell line .
five 
 transitions were monitored per peptide , as selected from the most 
 intense y - ions observed in a reference spectral library .
on the basis 
 of this initial screen , 4 peptides were selected that gave the greatest 
 signal intensity and a cv of less than 25% when analyzing 1 g 
 of tryptic digest .
to enable quantitation by stable isotope 
 dilution , labeled peptides 
 corresponding to these 4 sequences were obtained and the limit of 
 quantitation was determined in the absence of biological matrix .
limit 
 of quantitation was defined as the concentration of peptide where 
 the cv for triplicate measurements exceeded 25% .
the peptides dppfcvar 
 and elvsefsr provided the greatest sensitivity , yielding limits of 
 quantitation within a biologically relevant range of 0.33 and 0.1 
 fmol on - column , respectively ( supporting information 
 figure 2 ) .
these limits correspond to a lower limit of detection 
 of 12  000 her2 receptors per cell , assuming 200 pg protein 
 per cell and 1 g tryptic digest on - column ( i.e. , 5000 cell 
 equivalents injected for mrm analysis ) .
detection limits for peptides 
 gqecveecr and npqlcyqdtilwk exceeded 1 fmol on - column and were not 
 considered for quantitation .
we then analyzed ffpe and frozen 
 xenograft tissue samples from 
 human bt474 and sum159 ( her2-negative ) breast cancer cell lines . whereas 
 signals from her2 peptides were readily discernible in bt474 xenografts , 
 her2 peptide signals were absent in sum159 xenografts , consistent 
 with their low level of her2 expression ( figure 4 ) .
estimates of her2 receptor numbers from quantification of the 
 dppfcvar peptide were 60% higher than from quantification of the elvsefsr 
 peptide .
the dppfcvar peptide is derived from the ectodomain of the 
 her2 receptor , whereas elvsefsr is in the intracellular activation 
 domain of the receptor .
indeed , bt474 xenografts have 
 been reported to shed the her2 extracellular domain into the circulation .
mrm chromatograms for the her2 intracellular domain peptide 
 elvsefsr 
 from analyses of ffpe xenograft specimens derived from the her2-overexpressing 
 cell line bt474 ( left ) and the non - her2-expressing cell line sum159 
 ( right ) .
. monitored 
 mrm transitions are depicted for the unlabeled , endogenous peptide , 
 and the isotope - labeled internal standards ( insets ) .
the intensity 
 scales ( y - axis ) are identical in the two sets of 
 plots . to explore the application of mrm for analyses 
 of her2 in ffpe 
 human breast tumor tissues
, we analyzed 5 her2-overexpressing breast 
 cancer tissues and 5 her2-negative breast cancers , which had been 
 classified based on previous ihc analysis in the vanderbilt clinical 
 immunohistochemistry laboratory .
mrm analyses of these specimens ( figure 5b ) demonstrate a clear difference in signal intensity 
 for her2 peptides between the her2 + and her2 negative tumors and indicate 
 a wide range of biological variability in receptor expression levels . 

quantification of receptor levels was performed by stable isotope 
 dilution . assuming a yield of 200 pg protein from an average cell , 
 we estimated her2 receptor levels ranging from 110  000 to 468  000 
 receptors per cell in the her2 positive cancers and fewer than 14  000 
 receptors per cell in the her2 negative cancers . because of the higher 
 limit of quantification for the dppfcvar peptide , it was not possible 
 to use this peptide to quantify receptor levels in all specimens . 
 in tissues most highly overexpressing her2 ,
quantification of receptor 
 levels based on dppfcvar yielded the same result as elvsefsr quantification , 
 suggesting no evidence of receptor ectodomain shedding in the tissues 
 analyzed .
( a ) quantification of her2 receptor protein in frozen ( red bars ) 
 and ffpe ( blue bars ) bt474 xenograft tissues . her2 protein was quantified 
 based on mrm of peptides representing the extracellular ( dppfcvar ) 
 and intracellular ( elvsefsr ) domains of the receptor .
plotted values are mean  sd 
 for 3 process replicates of one frozen and one ffpe tumor .
( b ) quantification 
 of her2 receptor protein in 5 human her2-positive and 5 her2 negative 
 human ffpe breast tumor tissues .
mrm quantitation was by stable isotope 
 dilution analysis of peptides representing the extracellular ( dppfcvar ) 
 and intracellular ( elvsefsr ) domains of her2 .
the goal of this work was to assess the 
 performance characteristics 
 of protein quantitation by mrm in ffpe tissue and to apply mrm to 
 analyze a clinically relevant biomarker in human ffpe tissue specimens . 

our studies addressed the hypothesis that protein cross - linking in 
 ffpe tissues would add to variability in mrm measurements , particularly 
 for lysine c - terminal peptides . although peptide yields , particularly 
 for lysine c - terminal tryptic peptides , are lower in ffpe tissues 
 than in frozen tissues , measurement variation is not significantly 
 different between these specimen types .
median cvs for analyses of 
 tryptic peptides in ffpe and frozen tissues were all below 20% and 
 were not significantly different between specimen type or between 
 lysine- and arginine c - terminal peptides .
the data not only demonstrate 
 that mrm analyses can be performed with equal precision on ffpe and 
 frozen tissues , but also indicate that lysine c - terminal peptides 
 need not be excluded as mrm candidates when working with ffpe tissue . 

our analyses of her2 protein in ffpe breast tumor xenografts and in 
 human ffpe breast tissue specimens illustrated the feasibility of 
 applying mrm to quantify clinically important tissue biomarkers .
the major question regarding the use of proteomic methods to analyze 
 ffpe tissue is the impact of protein cross - linking that occurs during 
 formalin fixation .
the terms  antigen retrieval  and 
  crosslink reversal  have been applied to describe the 
 various protocols to prepare ffpe tissue for ihc analysis .
although these approaches facilitate ihc , there is no molecular 
 or chemical evidence to demonstrate that formaldehyde - derived cross - links 
 have been reversed . indeed , given the chemical stability of many formaldehyde - derived 
 covalent cross - links in proteins , it is unlikely 
 that any method or reagent could quantitatively reverse cross - links 
 while sparing peptide bonds and other functional groups .
our 
 results suggest the chemical modifications induced by formalin 
 fixation decrease the sensitivity of mrm measurements .
thus , somewhat 
 fewer targets are accessible to mrm when ffpe tissue is analyzed . 

the fractional loss of peptide signal is negligible for high - abundance 
 proteins , but proteins near the limit of quantitation in frozen tissue 
 are less likely to be accurately quantified by mrm in ffpe tissue . 

this limitation may be overcome to some extent through increased sample 
 input and possibly through the use of larger capacity chromatography 
 columns , permitting injection of larger amounts of tryptic digest . 

however , our data indicate that the overall impact of formalin fixation 
 on peptide yield and signals was significant , yet modest and does 
 not preclude application of mrm to analyze proteins in ffpe tissues . 

we should also point out that , despite the validity of this general 
 conclusion , certain peptides may be unusually sensitive to formalin 
 fixation and display high measurement variation in ffpe samples .
thus , 
 mrm assay development should consider multiple peptides during assay 
 development and optimization .
we were unable to assess the accuracy 
 of quantitation in ffpe tissues 
 because the concentrations of the proteins analyzed were not known . 

spike - in experiments with known amounts of target proteins to assess 
 quantitative accuracy are not possible with ffpe tissues , as the spiked 
 proteins would not control for the formalin fixation steps , which 
 probably have the greatest effect on protein yield .
assessment of 
 accuracy in mrm analyses of any sample type is complicated by uncontrolled 
 variability in protein digestion and recovery . however , the goal of mrm analyses usually is comparison 
 of protein levels , rather than assessment of absolute amounts and 
 the underlying assumption is that deviations from accuracy are evenly 
 distributed across all measurements .
precise and reproducible measurement 
 of relative differences between sample classes can enable biomarker 
 verification and validation studies . to evaluate mrm analyses 
 of ffpe tissues to quantify a clinically 
 relevant protein biomarker , we analyzed her2 receptor expression levels 
 in her2 positive and her2 negative breast tumors .
her2 is an important 
 diagnostic and predictive factor , as patients with her2-positive tumors 
 are candidates for anti - her2 therapies .
her2-positive tumors are defined 
 by intense membrane staining in the majority of tumor cells ( 3 + by 
 ihc ) using antibodies directed against the her2 c - terminus or by expression 
 of 2.2 copies of the her2 gene , as determined 
 by fluorescent in situ hybridization ( fish ) .
ihc staining or her2 copy number determination in ffpe tissue sections 
 is semiquantitative in nature and results are dependent on antibody 
 performance and operator experience .
further , the rate of discordance 
 between ihc and fish can approach 20% , which is significantly below 
 the maximum rate of 5% recommended by the american society of clinical 
 oncologists .
an mrm - based approach 
 to analysis of tissue biomarker proteins 
 offers potential advantages over ihc .
most significantly , mrm would 
 allow a systematic approach to configuration of targeted assays for 
 tissue proteins , even when antibody reagents validated for ihc are 
 unavailable .
this would remove a major barrier to analysis of protein 
 biomarkers in ffpe specimens .
mrm analyses also provide additional 
 molecular detail that may not be accessible through ihc .
for example , 
 analyses of her2 protein with mrm quantitation of both extracellular 
 and intracellular sequences can extend the specificity of molecular 
 characterization of her2-positive breast cancers . a potential mechanism 
 of resistance to the her2-targeted drug trastuzumab
is shedding of 
 the receptor ectodomain , as her2 is subject to proteolytic processing 
 at the plasma membrane by an adam or mmp .
her2 measurement based on mrm analysis of intracellular and extracellular 
 peptide sequences provides a more informative assessment of the status 
 of the her2 target than does ihc and fish .
mrm could further enable 
 analysis of modified or variant protein forms , which may be difficult 
 to detect selectively with antibody reagents .
our analyses of 
 her2 in ffpe tissue specimens illustrate the potential 
 utility of mrm for analysis of a clinically relevant tissue protein 
 biomarker .
the data demonstrate that mrm analyses are concordant with 
 her2 status as measured by ihc .
an assessment of the performance of 
 mrm for her2 or other tissue biomarkers is beyond the scope of this 
 preliminary study .
nevertheless , our results demonstrate that mrm 
 analyses in ffpe and frozen tissues display similar performance characteristics 
 and that measurement precision in the two tissue types is essentially 
 identical .
although target peptide yields are lower in ffpe tissues , 
 this has a modest effect on assay sensitivity .
our studies were limited 
 to measurements of unmodified peptides and may not be applicable to 
 post - translationally modified ( e.g. , phosphorylated , n - acetylated ) 
 sequences , where the stability of labile modifications during tissue 
 harvest and fixation may affect the results .
our findings indicate 
 that effects of formalin fixation on tissue proteins does not present 
 major potential barrier to the development of mrm - based tissue assays 
 for protein expression for research purposes .
the data also suggest 
 that further development of mrm instrumentation and approaches may 
 provide powerful new diagnostic tools for the surgical pathology laboratory .
 we compared the reproducibility of multiple reaction 
 monitoring 
 ( mrm ) mass spectrometry - based peptide quantitation in tryptic digests 
 from formalin - fixed , paraffin - embedded ( ffpe ) and frozen clear cell 
 renal cell carcinoma tissues . 
 the analyses targeted a candidate set 
 of 114 peptides previously identified in shotgun proteomic analyses , 
 of which 104 were detectable in ffpe and frozen tissue . although signal 
 intensities for mrm of peptides from ffpe tissue were on average 66% 
 of those in frozen tissue , median coefficients of variation ( cv ) for 
 measurements in ffpe and frozen tissues were nearly identical ( 1820% ) . 
 measurements of lysine c - terminal peptides and arginine c - terminal 
 peptides from ffpe tissue were similarly reproducible ( 19.5% and 18.3% 
 median cv , respectively ) . 
 we further evaluated the precision of mrm - based 
 quantitation by analysis of peptides from the her2 receptor in ffpe 
 and frozen tissues from a her2 overexpressing mouse xenograft model 
 of breast cancer and in human ffpe breast cancer specimens . 
 we obtained 
 equivalent mrm measurements of her2 receptor levels in ffpe and frozen 
 mouse xenografts derived from her2-overexpressing bt474 cells and 
 her2-negative sum159 cells . 
 mrm analyses of 5 her2-positive and 5 
 her - negative human ffpe breast tumors confirmed the results of immunohistochemical 
 analyses , thus demonstrating the feasibility of her2 protein quantification 
 in ffpe tissue specimens . 
 the data demonstrate that mrm analyses can 
 be performed with equal precision on ffpe and frozen tissues and that 
 lysine - containing peptides can be selected for quantitative comparisons , 
 despite the greater impact of formalin fixation on lysine residues . 
 
 the data further illustrate the feasibility of applying mrm to quantify 
 clinically important tissue biomarkers in ffpe specimens . 
Introduction
Experimental Procedures
Results
Discussion