proteomics has emerged as a valuable tool 
 for the identification 
 of biomarkers associated with human disease including those resulting from very subtle changes in normal cell 
 functions and signaling pathways . proteomic technology has advanced 
 to a state where thousands of proteins can be identified within complex 
 samples , yet disease - based studies using fresh or frozen tissues are limited 
 by a lack of available specimens for longitudinal clinical investigations . in contrast , there are millions of archival formalin - fixed , paraffin - embedded 
 ( ffpe ) tissues for which the clinical course of disease and response 
 to therapy has been established . ffpe tissue studies are affected 
 greatly by sample collection , tissue processing , and archival time . though these factors are difficult to control 
 in archival samples , improvements in techniques such as mtraq have 
 made relative quantitation of disease biomarkers in ffpe tissue possible . improving protein extraction and detection of less abundant protein 
 biomarkers is also of critical importance in proteomic analysis . protein 
 modifications by formaldehyde treatment and histological processing significantly limit the use ffpe tissues for proteomic analyses . this has prevented proteomic studies of the clinical course of diseases , 
 such as prostate and breast cancer , that evolve slowly or where the 
 time between treatment and recurrence is long . coupling proteomic 
 investigations with the retrospective pathology information available 
 from archival ffpe tissues would produce a wealth of practical information 
 on human diseases . some are practical for slide - mounted ffpe tissue , such as 
 quantitative fluorescence imaging analysis ( qfia ) , which is reproducible 
 and sensitive for specific standardized proteins , or maldi - imaging 
 mass spectrometry ( ms ) . other encouraging mass spectrometry 
 ( ms)-based proteomic studies of ffpe tissues have appeared in the 
 recent literature ; however , these investigations have typically been 
 restricted to minute tissue specimens , such as those obtained by laser 
 capture microdissection . further , some studies report high rates of 
 false - positive protein identification and are limited to the analysis 
 of tryptic digests of ffpe tissues by liquid chromatography  ms 
 ( lc  ms ) . our laboratory has been studying the reactions 
 of formaldehyde 
 with proteins and ways to reverse these reactions . using proteins in aqueous solution , we demonstrated that 
 the majority of protein formaldehyde adducts and cross - links were 
 consistently reversed with mild heating following the removal of excess 
 formaldehyde by dialysis . we then developed 
 a tissue surrogate , which consists of one or more proteins that form 
 a gel - like plug when treated with formaldehyde at protein concentrations 
 exceeding 75 mg / ml . these tissue surrogates have sufficient physical 
 integrity to be processed using normal histological methods . a variety of extraction buffers and heating 
 protocols were examined for their ability to recover proteins from 
 tissue surrogates . protein recovery was generally modest , and studies 
 with multiprotein tissue surrogates revealed extraction bias , meaning 
 that the composition of the solubilized proteins did not match that 
 of the corresponding tissue surrogate . subsequent studies showed that 
 the ethanol dehydration step of histology caused most formaldehyde - treated 
 proteins to adopt conformations enriched in -sheets , leading 
 to the formation of protein aggregates where the -sheets form 
 a dense network of intermolecular formaldehyde cross - links . we proposed that the difficulty of rehydrating 
 these stabilized protein aggregates was the primary impediment to 
 recovering proteins from ffpe tissue . pressure promotes water 
 penetration into the inner core of proteins , 
 causing denaturation , whereas heat alone causes protein unfolding 
 followed by aggregation . consequently , 
 we hypothesized that the combined effects of heat and elevated pressure 
 would facilitate the rehydration of the highly cross - linked protein 
 aggregates in ffpe tissues . the increased exposure to water should 
 greatly improve protein solubilization while simultaneously promoting 
 the reversal of protein  formaldehyde adducts and cross - links . initial physical studies on tissue surrogates supported this hypothesis and suggested that the effect of pressure was 
 to reduce the size of protein aggregates through increased water penetration , 
 rather than to increase the rate of reversal of protein  formaldehyde 
 adducts and cross - links directly . encouraged by these results , we extracted ffpe mouse liver tissues 
 with heat augmented by elevated hydrostatic pressure , with the goal 
 of reducing extraction bias , improving the recovery of intact proteins , 
 and obtaining tryptic digests that more closely resemble those from 
 matched fresh - frozen tissue . tissue tek oct compound was purchased from 
 sakura , usa , amicon ultra 3k centrifugal filters were purchased from 
 millipore , and 37% formaldehyde and pierce detergent removal columns 
 were obtained from thermo fisher . precast nupage bis - tris 412% 
 gels , 2-(n - morpholino)ethanesulfonic acid sds 
 running buffer , and the silverquest staining kit were purchased from 
 life technologies . tissue extracts were 
 heated under a pressure of 40,000 psi ( 276 mpa ) using both home - built 
 and commercial instruments . the home - built instrument consisted of 
 a 2-ml capacity ms-1 stainless steel reaction vessel coupled to a 
 manually operated high pressure piston screw pump available from high 
 pressure equipment company ( erie , pa ) . the temperature of the pressure 
 vessel was regulated by a eurotherm 2132 temperature controller ( leesburg , 
 va ) connected to an aluminum heating collar surrounding the reaction 
 vessel . the tissue extract ( 2 ml ) was added directly to the reaction 
 vessel using a syringe . the construction and operation of this pressure 
 system has been described in detail previously . the commercial instrument was a model nep 2320 barocycler ( pressure 
 biosciences ) modified by the manufacturer to hold isobaric pressure 
 and to provide temperature control up to 95 c . the tissue extract 
 ( 2 ml ) was added to a ft500 sample tube , which was capped and placed 
 in the pressure vessel of the barocycler . the liver from a female balb / c 
 mouse was obtained under the secondary use provision from the department 
 of laboratory animal medicine of the armed forces institute of pathology 
 ( usa ) . half of the liver was immediately frozen in tissue - tek oct 
 compound , divided into several equal - sized pieces , and stored at 80 
 c . the other half was fixed for 48 h at 4 c in 10% formalin . the fixed liver tissue was washed for 30 min with distilled water 
 and dehydrated through a graded series of alcohols ( 70 , 85 , and 100% 
 by volume ) and two changes of xylene , 30 min each . recovery experiments , 10 m sections of ffpe mouse liver were 
 cleared of paraffin by incubating the sections through two changes 
 of xylene for 10 min each . the sections were rehydrated through a 
 series of graded alcohols for 10 min each2 changes each of 
 100% ethanol , 85% ethanol , and 70% ethanol  and then incubated 
 in distilled water for a minimum of 30 min , as described previously . matched fresh - frozen and ffpe liver tissue sections 
 were analyzed after 30 days , and again after 1 year , of storage . the ability 
 of elevated hydrostatic pressure combined with heat to improve the 
 recovery of proteins from ffpe mouse liver tissue was evaluated using 
 two heat - based ffpe proteomic protocols recently reported in the literature . for each protocol , the experimental procedure was followed exactly 
 as published , but with the heating step divided into two arms , or 
 variations . in the first arm , the ffpe tissue extract was heated at 
 the temperature and for the length of time reported in the original 
 method . in the second experimental arm , the temperature and length 
 of time was identical , but the experiment was performed under a pressure 
 of 40,000 psi . the two experimental arms were then analyzed using 
 identical gel electrophoresis and lc / ms conditions . the protein content 
 of the tissue extracts was determined using a bca protein assay kit 
 ( pierce ) . following deparaffinization and 
 rehydration , the tissue was cut into small pieces and suspended in 
 36 ml of 50 mm tris - hcl , ph 7 , 2% sds ( extraction buffer 1 , 
 eb1 ) as described by shi et al . the tissue 
 suspension was homogenized with three 5-s cycles of sonication on 
 ice using a probe - tip sonicator , and the resulting homogenate was 
 split into two equal fractions . one fraction ( protocol-1a ) was incubated 
 at 100 c for 30 min followed by 80 c for 2 h at atmospheric 
 pressure ( 14.7 psi ) in a sand bath . the second fraction ( protocol-1p ) 
 was incubated at 100 c for 30 min followed by 80 c for 
 2 h at 40,000 psi using our hand - built pressure instrument . a 750 
 m section ( approximate thickness ) of matched fresh - frozen liver 
 was cut with a razor blade and homogenized in 22.5 ml of eb1 by sonication 
 as described above . proteins were extracted using two methods : incubation 
 of the homogenate in an ice bath for 2.5 h ( protocol-1fi ) or incubation 
 at 100 c for 30 min followed by 80 c for 2 h at atmospheric 
 pressure using a sand bath ( protocol-1fh ) . following deparaffinization and rehydration , the 
 tissue was cut into small pieces and suspended in 36 ml of 
 100 mm tris - hcl , ph 8 , 100 mm dtt , 4% sds ( extraction buffer 2 , eb2 ) 
 as described by ostasiewicz et al . and 
 homogenized with three 5-s cycles of sonication on ice using a probe - tip 
 sonicator . one fraction ( protocol-2a ) was incubated at 95 c 
 for 1 h at atmospheric pressure in a sand bath . the second fraction 
 ( protocol-2p ) was incubated at 95 c for 1 h at 40,000 psi using 
 the barocycler instrument . a 750 m section of matched fresh - frozen 
 liver was cut with a razor blade and homogenized in 22.5 ml of eb2 
 by sonication as described above . proteins were extracted by incubating 
 the homogenate at 95 c for 3 min at atmospheric pressure using 
 a sand bath ( protocol-2fh ) . all ffpe and matched 
 fresh - frozen mouse liver tissue extracts ( 40 g each ) were separated 
 by sds - page on precast nupage bis - tris 412% gels using 2-(n - morpholino)ethanesulfonic acid the gels were stained using the silverquest silver staining kit and 
 documented using an epson v500 photoscanner and annotated in adobe 
 photoshop , version 7.1 . each gel lane was then divided into 10 bands 
 and placed into microcentrifuge tubes . in - gel tryptic digestion was 
 carried out as previously described . separation of the 
 digested peptides was performed using nanocolumns prepared in - house 
 ( 75 m i.d . packed with jupiter c18 particles , 5 m , 300 
  ) connected to an agilent 1100 nanoflow lc system , which 
 was used to deliver binary gradient solvents a ( 0.1% formic acid ( fa ) 
 in water ) and b ( 0.1% fa in acetonitrile ) . reversed - phase chromatography 
 was performed by solubilizing the lyophilized tryptic peptides in 
 10 l of 0.1% trifluoroacetic acid and injecting 7 l 
 of sample per analysis . after sample injection , a 20-min wash with 
 98% mobile phase a was used to remove any remaining salts from the 
 sample . peptide elution was accomplished using a linear gradient of 
 2% solvent b to 42% solvent b over 45 min at a constant flow rate 
 of 250 nl / min . the nanoflow reversed - phase lc column was coupled 
 online to a linear ion trap mass spectrometer ( ltq , thermoelectron ) 
 using the manufacturer s nanoelectrospray source with an applied 
 electrospray potential of 1.75 kv and a capillary transfer tube temperature 
 of 185 c . the ltq - ms was operated in a data - dependent mode where 
 each full ms scan was followed by seven tandem ms scans in which the 
 seven most abundant peptide molecular ions detected were dynamically 
 selected for ms / ms analysis using a normalized cid energy of 35% . 
 a dynamic exclusion of 60-s was applied to reduce redundant selection 
 of peptides . the ms / ms spectra were analyzed using sequest ( thermoelectron ) 
 against a combined uniprot nonredundant mouse proteome database containing 
 36,799 protein sequences . only peptides with conventional tryptic 
 termini ( allowing for up to two internal missed cleavages ) possessing 
 delta - correlation scores ( cn ) > 0.08 and charge state - dependent cross - correlation ( xcorr ) criteria as follows were considered as 
 legitimate identifications : > 1.9 for + 1 charged peptides , > 2.2 
 for 
 + 2 charged peptides , and > 3.1 for + 3 charged peptides . a reverse - database 
 search , performed using the respective databases , resulted in a calculated 
 false - positive rate of < 2% for all samples analyzed . ffpe 
 and matched fresh - frozen mouse liver tissue stored for 30 days were 
 extracted using protocol-1 . the fresh liver tissue was completely 
 solubilized in the extraction buffer ( eb1 ) using extraction either 
 on ice ( protocol-1fi ) or at elevated temperature ( protocol-1fh ) . the 
 quantity of protein solubilized in the buffer was designated as 100% 
 protein recovery in table 1 . ffpe mouse liver 
 tissue extracted at atmospheric pressure ( protocol-1a ) resulted in 
 a protein recovery of only 17% , and a large plug of remaining tissue 
 was observed in the extraction vial . in contrast , when the extraction 
 was performed at 40,000 psi ( protocol-1p ) , the solubilized protein 
 increased to 77% and only a small amount of tissue residue remained 
 in the vial . ffpe and matching fresh - frozen liver tissue stored for 
 1 year were extracted using protocol-2 . the fresh liver tissue was 
 completely solubilized in the extraction buffer ( eb2 ) when extracted 
 at elevated temperature ( protocol-2fh ) . the quantity of protein solubilized 
 in the buffer was designated as 100% protein recovery in table 1 . the advantage of using elevated pressure was again 
 evident , as 79% of the ffpe liver protein was solubilized at elevated 
 hydrostatic pressure ( protocol-2p ) while only 18% was recovered at 
 ambient pressure ( protocol-2a ) . ffpe mouse liver was homogenized 
 in extraction buffer and heated with or without elevated pressure . fresh - frozen tissue was extracted either at atmospheric pressure using 
 the indicated extraction condition or on ice for 2.5 h. protocol used for protein extraction 
 ( see materials and methods ) . tissue was heated at 100 c 
 for 30 min ; then the temperature was lowered to 80 c for 2 h. the amount of protein extracted 
 from fresh frozen tissue was set to 100% . previous studies using ffpe tissue surrogates containing 
 several 
 proteins indicated that failure to completely 
 solubilize the tissue surrogate led to extraction bias , such that 
 the composition of the solubilized protein solution differed significantly 
 from that of the original surrogate . the extraction bias became less 
 significant as the percentage of total protein solubilized from the 
 tissue surrogate increased . thus , the 
 4-fold improvement in protein solubilization realized when the extraction 
 was performed at elevated pressure is likely to lead to a protein 
 extract that more accurately represents the composition of the original 
 ffpe tissue . further , the increased amount of recovered protein allows 
 a greater number of analytical techniques to be performed . while most 
 published proteomic studies of ffpe tissue analyze only a few thousand 
 cells from microdissected tissue , the use of elevated pressure has the advantage of improving protein 
 extraction from whole tissue sections . this ability is particularly 
 useful in instances where tissue microdissection is not practical 
 or when a more global proteomic analysis is desired . unbiased protein 
 extraction and standardized protocols are particularly important with 
 techniques such as reverse phase protein arrays ( rppas ) , where the 
 protein components of cell signaling pathways are quantified to direct 
 clinical treatment of cancer . the 
 30-day - old ffpe mouse liver extracted at 40,000 psi ( protocol-1p ) 
 exhibited a number of well resolved high and low molecular weight 
 protein bands by sds - page , corresponding to 87% of those seen 
 in the matched fresh - frozen tissue extracted on ice ( figure 1 , lanes 2 and 1 , respectively ) . the ffpe samples 
 extracted at ambient pressure ( protocol-1a ) contained relatively few 
 well - resolved protein bands equivalent to 25% of those seen 
 in frozen mouse liver ( figure 1 , lane 3 ) . similar 
 results were seen with 1-year - old ffpe mouse liver extracted at 40,000 
 psi ( protocol-2p ) with well resolved high and low molecular weight 
 protein bands corresponding to 74% of those seen in the matched fresh - frozen 
 tissue ( not shown ) . when the extraction was performed ambient pressure 
 ( protocol-2a ) this value 1d sds - page of fresh - frozen 
 and ffpe mouse liver extracts : lane 
 1 , fresh - frozen tissue ( protocol-1fi ) ; lane m , molecular weight marker ; 
 lane 2 , ffpe tissue extracted with heat at 40,000 psi ( protocol-1p ) ; 
 lane 3 , ffpe tissue extracted with heat alone ( protocol-1a ) . as top - down ms sequencing technology improves , 
 the ability to extract 
 and analyze intact proteins from ffpe tissue will become more important . top - down sequencing facilitates the measurement of combinations of 
 modifications , such as phosphorylation and glycosylation , and the 
 direct quantitation of specific protein isoforms and splice variants . few of these measurements are directly obtainable using bottom - up 
 proteomic approaches in which proteins are digested into peptides . 
 the ability to extract intact proteins from the seemingly inexhaustible 
 source of ffpe tissues will increase the diagnostic and prognostic 
 efficacy of proteomic - based biomarker discovery by allowing biomarker 
 validation using orthogonal methods such as western blotting , immunohistochemistry , 
 immunoassays , and structural and interaction proteomics . in this context , 
 the > 3-fold increase in the recovery of intact proteins from ffpe 
 tissue achieved by using elevated pressure represents a significant 
 breakthrough . ffpe 
 and matched fresh - frozen liver tissue stored for 30 days were extracted 
 using protocol-1 . ffpe tissue was extracted at ambient pressure ( protocol-1a ) 
 or 40,000 psi ( protocol-1p ) , while the matched fresh - frozen liver 
 tissue was extracted using protocol-1fi and protocol-1fh . the solubilized 
 proteins were separated by 1d - page , and each gel lane was excised , 
 digested with trypsin , desalted , and analyzed by lc - ms / ms . the total 
 unique peptide and protein identifications for each tissue type and 
 extraction condition are shown in table 1 . ffpe tissue extracted with heat alone resulted in the identification 
 of 5565 unique peptides and 3449 unique proteins . the addition of 
 elevated hydrostatic pressure significantly improved both the number 
 of unique peptides ( 9621 ) and proteins ( 5192 ) identified . the number 
 of proteins identified from the high pressure - extracted sample was 
 comparable to the number of unique proteins identified from fresh - frozen 
 tissue , which ranged from 4932 for tissue extracted on ice to 4451 
 for frozen tissue extracted with heat ( table 1 ) . the ms results for the 30-day - old ffpe mouse liver extracted 
 under elevated pressure ( protocol-1p ) and the matched fresh - frozen 
 tissue extracted on ice ( protocol-1fi ) were searched using gominer , 
 a gene ontology program . the percentages of nuclear , membrane , intracellular , 
 and extracellular proteins identified in fresh - frozen and ffpe liver 
 were virtually identical ( figure 2a ) , as were 
 the results for classification by biological function ( figure 2b ) . gene ontology analysis of proteins identified by lc - ms / ms . 
 proteins 
 identified using fresh - frozen mouse liver ( protocol-1fi ) or ffpe liver 
 extracted with heat and elevated pressure ( protocol-1p ) were categorized 
 by subcellular localization ( a ) or biological process ( b ) , using gominer 
 gene ontology software . to address the effect of long - term storage of the 
 ffpe specimens , 
 the mouse liver samples were investigated after an additional 11 months 
 of storage ( 1-year - old sample ) . ffpe tissue was extracted at ambient 
 pressure ( protocol-2a ) or 40,000 psi ( protocol-2p ) , while the matched 
 fresh - frozen liver tissue was extracted at high temperature ( protocol-2fh ) . the solubilized proteins were separated by 1d - page , and each gel lane 
 was excised , digested with trypsin , desalted , and analyzed by lc - ms / ms . 
 from the 1-d gel , we were able to identify 3492 nonredundant proteins 
 in the 1-year - old ffpe liver extracted under elevated pressure , which 
 was comparable to the 3415 nonredundant proteins identified in the 
 matched fresh - frozen mouse liver extracted at high temperature . in 
 contrast , only 107 unique proteins were identified in the ffpe tissue 
 extracted at ambient pressure . figure 3 shows venn diagrams of the unique 
 and common ( overlapping ) proteins identified in mouse liver tissue 
 extracts prepared by different methods . figure 3a compares 30-day - old fresh - frozen liver tissue extracted on ice 
 ( protocol-1fi ) versus 100 c for 30 min followed by 80 c 
 for 2 h ( protocol-1fh ) . figure 3b compares 
 30-day - old ffpe liver tissue extracted under pressure ( protocol-1p ) figure 3c compares 1-year - old 
 ffpe liver tissue extracted under pressure ( protocol-2p ) versus matched 
 fresh - frozen liver extracted at elevated temperature ( protocol-2fh ) . notably , the common proteins , expressed as a percentage of either 
 the ffpe or matched fresh - frozen mouse liver tissue , were 50% 
 for all three tissue pairs . venn diagrams showing the number of unique and 
 common proteins 
 identified using lc ms / ms analysis : panel a , fresh - frozen tissue extracted 
 on ice ( protocol-1fi ) or with heat ( protocol-1fh ) ; panel b , fresh - frozen 
 tissue extracted with heat ( protocol-1fh ) and ffpe mouse liver extracted 
 with elevated pressure ( protocol-1p ) ; panel c , fresh - frozen tissue 
 extracted with heat ( protocol-2fh ) and ffpe mouse liver extracted 
 with elevated pressure ( protocol-2p ) . figure 4a is a pie chart 
 showing the number 
 of unique proteins identified by two or more fully tryptic peptides 
 for 30-day - old ffpe mouse liver extracted under pressure ( protocol-1p ) . the results reveal that 49% of the proteins were identified by two 
 peptides , 51% were identified by three or more peptides , and 21% of 
 the proteins were identified by five or more peptides . figure 4b shows similar results for 1-year - old ffpe mouse 
 liver extracted under pressure ( protocol-2p ) , with 57% of the proteins 
 identified by two peptides , 41% by three or more peptides , and 15% 
 identified by five or more peptides . these results are similar to 
 those for the matched fresh - frozen mouse liver tissue ( not shown ) . the complete list of peptides identified in the fresh - frozen and high - pressure - recovered 
 ffpe tissue , with their corresponding xcorr values , can be found online 
 ( supporting information table 1 ) . total number 
 of unique proteins identified using lc - ms / ms by two 
 or more unique , fully tryptic peptides in ffpe mouse tissue extracted 
 with heat and elevated pressure ( 40,000 psi ) : a , 30-day - old ffpe mouse 
 liver ( protocol-1p ) ; b , 1-year - old ffpe mouse liver ( protocol-2p ) . virtually all protocols reported in the 
 literature for the extraction 
 of proteins from ffpe tissue use a variation of the heat - induced antigen 
 retrieval technique developed by shi and taylor for the recovery of antigenicity in immunohistochemical 
 studies . this method involves exposing ffpe tissue sections to a buffer 
 solution containing a detergent and/or protein denaturant and elevated 
 temperatures of 90120 c for a period of 1030 
 min . optimal antigen recovery varies 
 with the host ffpe tissue , with each type requiring different buffers , 
 ph values , buffer additives , and incubation temperatures . although the ffpe proteomic literature is still quite limited , it 
 is not unreasonable to propose that the same situation applies to 
 the proteomic analysis of ffpe tissues . elevated hydrostatic pressure 
 is not a technique unto itself but rather an adjuvant method that 
 can be applied to existing or future ffpe protein extraction protocols . 
 while it remains to be shown that this method is useful with every 
 tissue fixation and protein extraction protocol , elevated pressure 
 acts through purely physical means and 
 should be compatible with ffpe protein extraction buffers of any ph 
 and containing any detergent , protein denaturant , or other additive . this should allow its integration into a wide 
 range of protein extraction protocols for ms - based proteomics with 
 little to no alteration to downstream sample preparation and analysis . this was demonstrated in this report by the successful application 
 of elevated pressure to two very different published ffpe protein 
 extraction protocols . an increase in pressure to 40,000 psi , to augment 
 heat treatment , improved protein extraction efficiency from ffpe mouse 
 liver tissue by approximately 4-fold and increased the number of unique 
 proteins identified by up to 30-fold over the published methods used 
 at ambient pressure . further , the tryptic digests of these pressure - extracted 
 tissues resulted in protein profiles that more closely resembled those 
 from matched fresh - frozen tissue when analyzed by lc / ms than did those 
 extracted with heat alone , while maintaining a false - identification 
 rate of < 2% . the ability of elevated pressure to significantly 
 improve the recovery of intact proteins from ffpe tissues over the 
 use of heat alone has great potential for broad application to top - down 
 proteomic studies for the identification of disease biomarkers .
formaldehyde - fixed , paraffin - embedded ( ffpe ) tissue repositories 
 represent a valuable resource for the retrospective study of disease 
 progression and response to therapy . 
 however , the proteomic analysis 
 of ffpe tissues has been hampered by formaldehyde - induced protein 
 modifications , which reduce protein extraction efficiency and may 
 lead to protein misidentification . here , we demonstrate the use of 
 heat augmented with high hydrostatic pressure ( 40,000 psi ) as a novel 
 method for the recovery of intact proteins from ffpe mouse liver . 
 when ffpe mouse liver was extracted using heat and elevated pressure , 
 there was a 4-fold increase in protein extraction efficiency , a 3-fold 
 increase in the extraction of intact proteins , and up to a 30-fold 
 increase in the number of nonredundant proteins identified by mass 
 spectrometry , compared to matched tissue extracted with heat alone . 
 more importantly , the number of nonredundant proteins identified in 
 the ffpe tissue was nearly identical to that of matched fresh - frozen 
 tissue .