BACKGROUND
formalin − fixation, paraffin − embedding  is routinely used to preserve human tissue samples for routine pathological diagnostics.
comprehensive archives of ffpe specimens have been established in most large institutes of pathology, harboring tissue specimens from most human diseases, including comparably rare medical conditions.
as these specimens are carefully annotated regarding diagnosis, treatment and patient outcome, they are a unique research resource for proteomic analysis aiming to identify key proteins in disease progression and treatment response.
in recent years considerable advances have been made regarding the proteomic analysis of ffpe specimens.
upon formalin fixation, extensive protein cross-linking occurs, largely involving the ε–amino group of lysine residues.
these covalent cross-links are reversed by sample heating in the presence of a strong detergent  or a chaotropic salt .
ffpe samples that were > 5 years old have been successfully analyzed by liquid chromatography  − tandem mass spectrometry  .
there have been considerable improvements with regard to proteome coverage  <cit>  and the ability to analyse post–translational modifications such as glycosylation or phosphorylation  <cit> .
quantitative proteomic analysis of ffpe specimens has been performed using two–dimensional differential gel electrophoresis  <cit>  or label–free chromatographic approaches  <cit> .
chemical isotope tagging of ffpe samples has been performed using itraq labeling .
reductive dimethylation of primary amines is a widely used strategy for relative quantification of peptides and proteins by lc-ms/ms  <cit> .
distinguishing features of dimethyl labeling are its robustness and cost–effectiveness together with the ability of binary and triplex approaches  <cit> .
it has recently been shown that quantification with dimethyl labeling is as accurate as metabolic labeling strategies  <cit> , which have been referred to as the gold standard in quantitative proteomics  <cit> .
in this study we establish the usage of dimethyl labeling for the quantitative proteomic investigation of ffpe specimens.
using corresponding cryopreserved tissue specimens as controls, we show that formalin fixation and paraffin embedding does not interfere with the formaldehyde–based dimethyl labeling.
in an exemplary proof–of–concept application, we used dimethyl labeling to profile proteome changes in ffpe tissue specimens of clear cell renal cell carcinoma , comparing ccrcc to adjacent non–malignant renal cells.
we found elevated levels of glycolytic enzymes that are in line with previous studies.
our findings add robust and cost–effective dimethyl labeling to the toolbox for quantitative proteomic analysis of ffpe specimens as well as providing further proteomic insight into ccrcc pathology.
RESULTS
overview
the present study aims to evaluate isotopic dimethyl labeling for the quantitative proteomic analysis of ffpe tissue specimens.
this stable isotope tagging method is then applied for quantitative proteome profiling of ccrcc tissue in comparison to adjacent non-malignant tissue.
labeling efficiency
we initially profiled ffpe–derived protein samples for the occurrence of dimethylated primary amines .
for ffpe samples and cryopreserved non-labeled control protein samples, we did not detect relevant levels of mono– or dimethylated peptides n-termini  or lysine side chains .
several reports indicate that ffpe protein samples are susceptible to a +12 da addition at n-termini as well as lysine, tryptophan, and tyrosine residues .
using the non-dimethylated protein samples, we probed for occurrence of these modifications.
for both ffpe and cryopreserved samples, less than 5 % of the identified peptides showed these modifications .fig.
<dig> modifications present at n-termini and lysine side chains before and after reductive dimethylation with either light  or heavy  formaldehyde.
data represents average values ± standard deviation; based on the analysis of three proteome samples from solid tumors.
identification numbers  are stated.
a) modifications of peptide n-termini before di-methylation; b) modifications of lysine side-chains before dimethylation; c) modifications of peptide n-termini after di-methylation; d) modifications of lysine side-chains after di-methylation
next, we assessed whether ffpe–derived protein samples are amenable for dimethyl labeling on the peptide level after trypsination.
to discriminate between the chemically introduced label and arbitrarily introduced modifications , we used heavy formaldehyde .
for both ffpe samples and cryopreserved control samples, dimethylation works robustly as > 95 % of all identified peptides were present in the heavy dimethylated form with regard to their n–termini  as well as lysine side chains .
we did not detect relevant levels of unlabeled, monomethylated or light-dimethylated peptides.
protein samples derived from both ffpe or cryopreserved control tissue specimens yield comparable peptide identification numbers, which are in the range of recent reports of proteomic analysis of ffpe tissues  <cit> .
together, these results highlight that ffpe specimens are amenable to dimethyl labeling without interference from the initial formalin fixation.
quantitation accuracy
to investigate whether dimethyl labeling yields accurate quantification of ffpe –derived protein samples, we halved ffpe samples after trypsination, labeled the different aliquots with either light  or heavy  formaldehyde, and mixed the aliquots at a 1: <dig> ratio, followed by lc–ms/ms analysis .
the same procedure was conducted with cryopreserved control samples .
the analysis was performed in triplicate, yielding consistently between  <dig> and  <dig> protein identifications.
the fc-values  display a narrow distribution, with almost identical standard deviations, ranging from  <dig>  –  <dig>  for the cryopreserved samples and from  <dig>  –  <dig>  for the ffpe samples.
a recent benchmarking study of dimethyl labeling showed a standard deviation of  <dig>  for a 1: <dig>  mix of an identical sample that was differentially labeled using dimethylation  <cit> .
our results are in very good agreement with the previously reported outcome.
recently, wakabayashi et al.
have also shown applicability of reductive dimethylation to ffpe-extracted phosphopeptides  <cit> .fig.
<dig> quantitative proteomic comparison of identical samples that were split, labeled by reductive dimethylation with either light  or heavy  formaldehyde, and mixed 1: <dig>  box-and-whisker plots denote the 25– <dig> percentile and the  <dig> –  <dig> percentile, respectively.
data represents average values ± standard deviation; based on the analysis of three proteome samples from solid tumors.
protein and peptide identification numbers are  <dig> proteins/ <dig> unique peptides for cryo-sample 1;  <dig> proteins/ <dig> unique peptides for cryo-sample 2;  <dig> proteins/ <dig> unique peptides for cryo-sample 3;  <dig> proteins/ <dig> unique peptide for ffpe-sample 1;  <dig> proteins/ <dig> unique peptides for ffpe-sample 2;  <dig> proteins/ <dig> unique peptides for ffpe sample 3
in summary, ffpe tissue specimens are amenable to protein extraction and subsequent relative quantitation by isotopic dimethyl labeling in lc-ms/ms analysis.
application to quantitative proteome profiling of clear cell renal cell carcinoma
clear cell renal cell carcinoma  is among the ten most common human malignancies and accounts for more than 90 % of renal neoplasms.
as it is clinically occult in many patients, diagnosis often occurs only at a later stage of disease and approximately one third of patients presents with metastatic disease upon diagnosis.
response rates to treatment are generally low for metastatic ccrcc, leading to a median survival of less than one year  <cit> .
as a first application of isotopic dimethyl labeling for relative protein quantitation, we focused on ccrcc.
four cases of ccrcc were analyzed, in which tumor and adjacent normal tissue was represented in the same ffpe tissue blocks.
in accordance with the workflow established in this study, we used “light” and “heavy” dimethylation with isotopic formaldehyde for quantitative proteomic comparison of the protein samples derived from the malignant and non–malignant ffpe specimen areas.
protein samples of each case were analyzed separately.
to increase proteome coverage, we employed scx prefractionation.
the fc-values of each replicate experiment followed a near-normal distribution .
peptides with unlabeled n-termini or lysine side-chains constitute less than 3 % of all identified peptides .
as expected, the fc-values are broadly distributed in each replicate , indicating substantial proteome differences between ccrcc tissue and adjacent, non-malignant tissue.
we used the apex method to calculate protein abundances  <cit> .
the resulting apex scores displayed good correlation between the different replicates .fig.
<dig> quantitative proteomic comparison of ffpe derived ccrcc tumor tissue with ffpe derived, adjacent non-malignant tissue.
stable isotope labeling was achieved by reductive dimethylation with either light  or heavy  formaldehyde.
a box-and-whisker plots denoting the 25– <dig> percentile and the  <dig> –  <dig> percentiles, respectively, of the four replicates  correlation of apex scores  <cit> , showing the pearson correlation.
protein and peptide identification numbers are  <dig> proteins/ <dig> unique peptides for replicate 1;  <dig> proteins/ <dig> unique peptides for replicate 2;  <dig> proteins/ <dig> peptides for replicate 3;  <dig> proteins/ <dig> peptides for replicate 4
a total of  <dig> non-redundant proteins were identified,  <dig> of these were found in at least three replicates.
as previously described, we employed the following criteria to distinguish significantly affected proteins:  identification in at least three replicate experiments,  protein abundance differences resulted in a p-value <  <dig>  ,  protein abundance increased or decreased in with an average fc-value >  <dig>  or < − <dig>  .
with these criteria,  <dig> proteins were found to be increased in ccrcc tissue  whereas  <dig> proteins were found to be decreased in ccrcc tissue in comparison to adjacent non-malignant tissue .
the online search tool for the retrieval of interacting genes  was used to display connections between proteins with either elevated or decreased levels  <cit> .
string visualized several functional clusters for proteins that were found to be elevated in ccrcc , including ribosomal and proteasomal proteins as well as proteins involved in glycolysis and energy metabolism.fig.
<dig> string protein functional association network  <cit>  of proteins that were found to be significantly upregulated in ccrcc compared to adjacent non-malignant tissue .
string was employed using “high confidence”.
disconnected nodes are not shown.
connections are shown using standard string coloring scheme as highlighted in the legend
our finding of increased levels of proteins involved in glycolysis in ccrcc, compared to corresponding non-malignant kidney tissue is in line with further reports, including proteomic, metabolomic, and functionally genomic approaches; all of which point towards increased aerobic glycolysis  in ccrcc .
although not highlighted by a string cluster, we noticed elevated levels of annexins a <dig> and a <dig>  this is corroborated by further proteomic studies on ccrcc  <cit> .
for annexins ii and iv, tumor promoting roles in ccrcc have been suggested, based on in vitro findings indicating that annexin ii and iv, respectively, sustain tumor cell migration  <cit> .
our proteomic analysis of ccrcc further suggests elevated levels of ribosomal and proteasomal proteins, putatively indicating a generally increased protein turnover in ccrcc as compared to non-malignant kidney tissue.
previous proteomic analyses of ccrcc have not highlighted a ribosomal/proteasomal fingerprint.
there are however several reports that link proteasomal function to ccrcc.
for example, exome sequencing has previously shown that components of the ubiquitin system are frequently mutated in ccrcc  <cit> .
inactivation of the nuclear deubiquitinating enzyme bap <dig> is also a frequent event in ccrcc  <cit> .
moreover, increased serum levels of the 20 s proteasome have been found in ccrcc patients  <cit> .
we identified  <dig> proteasomal proteins.
all of these were increased in ccrcc with the exceptions of proteasome subunit β type- <dig> and type- <dig>   <dig> proteasomal proteins met our criteria for significantly increased abundance.
likewise, we identified  <dig> ribosomal or ribosome-associated proteins excluding mitochondrial ribosomal proteins.
all of these were increased in ccrcc with the exceptions of ribosome-binding protein  <dig> and 60s ribosomal protein l <dig>   <dig> proteasomal proteins met our criteria for significantly increased abundance.
in summary, application of isotopic dimethyl tagging allowed for the quantitative profiling of ccrcc tissue.
our analysis corroborated previously established proteome motifs of ccrcc, i.e.
aerobic glycolysis, as well as pointing to newly discovered proteome alterations, i.e.
increased levels of both ribosomal and proteasomal proteins.
our exemplary application focuses on a pairwise comparison of different samples.
dimethyl labeling is typically restricted to pairwise or triple comparisons.
we consider this setting to be its typical application.
this limitation can be overcome by using a differentially dimethylated standard sample, analogous to the super-silac strategy  <cit> .
by comparing multiple samples against this standard, a larger number of samples can be probed.
CONCLUSIONS
our results show that dimethyl labeling is applicable for the quantitative proteomic analysis of ffpe tissue specimens without interference from the formalin fixation process.
quantitation accuracy is comparable to cryopreserved tissue specimens.
an initial application of dimethyl labeling to ffpe specimens portrayed differences in the proteome composition of ccrcc compared to adjacent non–malignant tissue.
dimethyl labeling with isotopic formaldehyde is a robust and cost–effective labeling strategy for quantitative proteomics.
the present work adds dimethyl labeling to the toolbox for quantitative proteome analysis of ffpe specimens.

 formalin − fixation, paraffin − embedding is routinely used to preserve human tissue samples for routine pathological diagnostics. comprehensive archives of ffpe specimens have been established in most large institutes of pathology, harboring tissue specimens from most human diseases, including comparably rare medical conditions. as these specimens are carefully annotated regarding diagnosis, treatment and patient outcome, they are a unique research resource for proteomic analysis aiming to identify key proteins in disease progression and treatment response. in recent years considerable advances have been made regarding the proteomic analysis of ffpe specimens. upon formalin fixation, extensive protein cross-linking occurs, largely involving the ε–amino group of lysine residues. these covalent cross-links are reversed by sample heating in the presence of a strong detergent or a chaotropic salt. ffpe samples that were > 5 years old have been successfully analyzed by liquid chromatography  − tandem mass spectrometry. there have been considerable improvements with regard to proteome coverage and the ability to analyse post–translational modifications such as glycosylation or phosphorylation. quantitative proteomic analysis of ffpe specimens has been performed using two–dimensional differential gel electrophoresis or label–free chromatographic approaches. chemical isotope tagging of ffpe samples has been performed using itraq labeling. reductive dimethylation of primary amines is a widely used strategy for relative quantification of peptides and proteins by lc-ms/ms. distinguishing features of dimethyl labeling are its robustness and cost–effectiveness together with the ability of binary and triplex approaches. it has recently been shown that quantification with dimethyl labeling is as accurate as metabolic labeling strategies, which have been referred to as the gold standard in quantitative proteomics. in this study we establish the usage of dimethyl labeling for the quantitative proteomic investigation of ffpe specimens. using corresponding cryopreserved tissue specimens as controls, we show that formalin fixation and paraffin embedding does not interfere with the formaldehyde–based dimethyl labeling. in an exemplary proof–of–concept application, we used dimethyl labeling to profile proteome changes in ffpe tissue specimens of clear cell renal cell carcinoma, comparing ccrcc to adjacent non–malignant renal cells. we found elevated levels of glycolytic enzymes that are in line with previous studies. our findings add robust and cost–effective dimethyl labeling to the toolbox for quantitative proteomic analysis of ffpe specimens as well as providing further proteomic insight into ccrcc pathology.  overview the present study aims to evaluate isotopic dimethyl labeling for the quantitative proteomic analysis of ffpe tissue specimens. this stable isotope tagging method is then applied for quantitative proteome profiling of ccrcc tissue in comparison to adjacent non-malignant tissue. labeling efficiency we initially profiled ffpe–derived protein samples for the occurrence of dimethylated primary amines. for ffpe samples and cryopreserved non-labeled control protein samples, we did not detect relevant levels of mono– or dimethylated peptides n-termini or lysine side chains. several reports indicate that ffpe protein samples are susceptible to a +12 da addition at n-termini as well as lysine, tryptophan, and tyrosine residues. using the non-dimethylated protein samples, we probed for occurrence of these modifications. for both ffpe and cryopreserved samples, less than 5 % of the identified peptides showed these modifications.fig. modifications present at n-termini and lysine side chains before and after reductive dimethylation with either light or heavy formaldehyde. data represents average values ± standard deviation; based on the analysis of three proteome samples from solid tumors. identification numbers are stated. a) modifications of peptide n-termini before di-methylation; b) modifications of lysine side-chains before dimethylation; c) modifications of peptide n-termini after di-methylation; d) modifications of lysine side-chains after di-methylation next, we assessed whether ffpe–derived protein samples are amenable for dimethyl labeling on the peptide level after trypsination. to discriminate between the chemically introduced label and arbitrarily introduced modifications, we used heavy formaldehyde. for both ffpe samples and cryopreserved control samples, dimethylation works robustly as > 95 % of all identified peptides were present in the heavy dimethylated form with regard to their n–termini as well as lysine side chains. we did not detect relevant levels of unlabeled, monomethylated or light-dimethylated peptides. protein samples derived from both ffpe or cryopreserved control tissue specimens yield comparable peptide identification numbers, which are in the range of recent reports of proteomic analysis of ffpe tissues. together, these results highlight that ffpe specimens are amenable to dimethyl labeling without interference from the initial formalin fixation. quantitation accuracy to investigate whether dimethyl labeling yields accurate quantification of ffpe –derived protein samples, we halved ffpe samples after trypsination, labeled the different aliquots with either light or heavy formaldehyde, and mixed the aliquots at a 1: ratio, followed by lc–ms/ms analysis. the same procedure was conducted with cryopreserved control samples. the analysis was performed in triplicate, yielding consistently between and protein identifications. the fc-values display a narrow distribution, with almost identical standard deviations, ranging from – for the cryopreserved samples and from – for the ffpe samples. a recent benchmarking study of dimethyl labeling showed a standard deviation of for a 1: mix of an identical sample that was differentially labeled using dimethylation. our results are in very good agreement with the previously reported outcome. recently, wakabayashi et al. have also shown applicability of reductive dimethylation to ffpe-extracted phosphopeptides.fig. quantitative proteomic comparison of identical samples that were split, labeled by reductive dimethylation with either light or heavy formaldehyde, and mixed 1: box-and-whisker plots denote the 25– percentile and the – percentile, respectively. data represents average values ± standard deviation; based on the analysis of three proteome samples from solid tumors. protein and peptide identification numbers are proteins/ unique peptides for cryo-sample 1; proteins/ unique peptides for cryo-sample 2; proteins/ unique peptides for cryo-sample 3; proteins/ unique peptide for ffpe-sample 1; proteins/ unique peptides for ffpe-sample 2; proteins/ unique peptides for ffpe sample 3 in summary, ffpe tissue specimens are amenable to protein extraction and subsequent relative quantitation by isotopic dimethyl labeling in lc-ms/ms analysis. application to quantitative proteome profiling of clear cell renal cell carcinoma clear cell renal cell carcinoma is among the ten most common human malignancies and accounts for more than 90 % of renal neoplasms. as it is clinically occult in many patients, diagnosis often occurs only at a later stage of disease and approximately one third of patients presents with metastatic disease upon diagnosis. response rates to treatment are generally low for metastatic ccrcc, leading to a median survival of less than one year. as a first application of isotopic dimethyl labeling for relative protein quantitation, we focused on ccrcc. four cases of ccrcc were analyzed, in which tumor and adjacent normal tissue was represented in the same ffpe tissue blocks. in accordance with the workflow established in this study, we used “light” and “heavy” dimethylation with isotopic formaldehyde for quantitative proteomic comparison of the protein samples derived from the malignant and non–malignant ffpe specimen areas. protein samples of each case were analyzed separately. to increase proteome coverage, we employed scx prefractionation. the fc-values of each replicate experiment followed a near-normal distribution. peptides with unlabeled n-termini or lysine side-chains constitute less than 3 % of all identified peptides. as expected, the fc-values are broadly distributed in each replicate, indicating substantial proteome differences between ccrcc tissue and adjacent, non-malignant tissue. we used the apex method to calculate protein abundances. the resulting apex scores displayed good correlation between the different replicates.fig. quantitative proteomic comparison of ffpe derived ccrcc tumor tissue with ffpe derived, adjacent non-malignant tissue. stable isotope labeling was achieved by reductive dimethylation with either light or heavy formaldehyde. a box-and-whisker plots denoting the 25– percentile and the – percentiles, respectively, of the four replicates correlation of apex scores, showing the pearson correlation. protein and peptide identification numbers are proteins/ unique peptides for replicate 1; proteins/ unique peptides for replicate 2; proteins/ peptides for replicate 3; proteins/ peptides for replicate 4 a total of non-redundant proteins were identified, of these were found in at least three replicates. as previously described, we employed the following criteria to distinguish significantly affected proteins: identification in at least three replicate experiments, protein abundance differences resulted in a p-value < , protein abundance increased or decreased in with an average fc-value >  or < −. with these criteria, proteins were found to be increased in ccrcc tissue whereas proteins were found to be decreased in ccrcc tissue in comparison to adjacent non-malignant tissue. the online search tool for the retrieval of interacting genes was used to display connections between proteins with either elevated or decreased levels. string visualized several functional clusters for proteins that were found to be elevated in ccrcc, including ribosomal and proteasomal proteins as well as proteins involved in glycolysis and energy metabolism.fig. string protein functional association network of proteins that were found to be significantly upregulated in ccrcc compared to adjacent non-malignant tissue. string was employed using “high confidence”. disconnected nodes are not shown. connections are shown using standard string coloring scheme as highlighted in the legend our finding of increased levels of proteins involved in glycolysis in ccrcc, compared to corresponding non-malignant kidney tissue is in line with further reports, including proteomic, metabolomic, and functionally genomic approaches; all of which point towards increased aerobic glycolysis in ccrcc. although not highlighted by a string cluster, we noticed elevated levels of annexins a and a this is corroborated by further proteomic studies on ccrcc. for annexins ii and iv, tumor promoting roles in ccrcc have been suggested, based on in vitro findings indicating that annexin ii and iv, respectively, sustain tumor cell migration. our proteomic analysis of ccrcc further suggests elevated levels of ribosomal and proteasomal proteins, putatively indicating a generally increased protein turnover in ccrcc as compared to non-malignant kidney tissue. previous proteomic analyses of ccrcc have not highlighted a ribosomal/proteasomal fingerprint. there are however several reports that link proteasomal function to ccrcc. for example, exome sequencing has previously shown that components of the ubiquitin system are frequently mutated in ccrcc. inactivation of the nuclear deubiquitinating enzyme bap is also a frequent event in ccrcc. moreover, increased serum levels of the 20 s proteasome have been found in ccrcc patients. we identified proteasomal proteins. all of these were increased in ccrcc with the exceptions of proteasome subunit β type- and type- proteasomal proteins met our criteria for significantly increased abundance. likewise, we identified ribosomal or ribosome-associated proteins excluding mitochondrial ribosomal proteins. all of these were increased in ccrcc with the exceptions of ribosome-binding protein and 60s ribosomal protein l proteasomal proteins met our criteria for significantly increased abundance. in summary, application of isotopic dimethyl tagging allowed for the quantitative profiling of ccrcc tissue. our analysis corroborated previously established proteome motifs of ccrcc, i.e. aerobic glycolysis, as well as pointing to newly discovered proteome alterations, i.e. increased levels of both ribosomal and proteasomal proteins. our exemplary application focuses on a pairwise comparison of different samples. dimethyl labeling is typically restricted to pairwise or triple comparisons. we consider this setting to be its typical application. this limitation can be overcome by using a differentially dimethylated standard sample, analogous to the super-silac strategy. by comparing multiple samples against this standard, a larger number of samples can be probed.  our results show that dimethyl labeling is applicable for the quantitative proteomic analysis of ffpe tissue specimens without interference from the formalin fixation process. quantitation accuracy is comparable to cryopreserved tissue specimens. an initial application of dimethyl labeling to ffpe specimens portrayed differences in the proteome composition of ccrcc compared to adjacent non–malignant tissue. dimethyl labeling with isotopic formaldehyde is a robust and cost–effective labeling strategy for quantitative proteomics. the present work adds dimethyl labeling to the toolbox for quantitative proteome analysis of ffpe specimens. 
in quantitative proteomic analysis, reductive di-methylation of primary amines using stable isotopic formaldehyde variants is increasingly used due to its robustness and cost-effectiveness. in the present study we show for the first time that isotopic amine dimethylation can be used in a straightforward manner for the quantitative proteomic analysis of ffpe specimens without interference from formalin employed in the ffpe process. our study establishes isotopic amine dimethylation as a versatile tool for quantitative proteomic analysis of ffpe specimens and underlines proteome alterations in ccrcc. isotopic amine dimethylation of ffpe specimens showed equal labeling efficiency as for cryopreserved specimens. in an initial application, we profiled proteome changes in clear cell renal cell carcinoma ffpe tissue specimens compared to adjacent non–malignant renal tissue. for both ffpe and cryopreserved specimens, differential labeling of identical samples yielded highly similar ratio distributions within the expected range for dimethyl labeling. our findings highlight increased levels of glyocolytic enzymes, annexins as well as ribosomal and proteasomal proteins.
 formalin-fixed, paraffin-embedded tissues represent the most abundant resource of archived human specimens in pathology. such tissue specimens are emerging as a highly valuable resource for translational proteomic studies. in quantitative proteomic analysis, reductive di-methylation of primary amines using stable isotopic formaldehyde variants is increasingly used due to its robustness and cost-effectiveness.  in the present study we show for the first time that isotopic amine dimethylation can be used in a straightforward manner for the quantitative proteomic analysis of ffpe specimens without interference from formalin employed in the ffpe process. isotopic amine dimethylation of ffpe specimens showed equal labeling efficiency as for cryopreserved specimens. for both ffpe and cryopreserved specimens, differential labeling of identical samples yielded highly similar ratio distributions within the expected range for dimethyl labeling. in an initial application, we profiled proteome changes in clear cell renal cell carcinoma ffpe tissue specimens compared to adjacent non–malignant renal tissue. our findings highlight increased levels of glyocolytic enzymes, annexins as well as ribosomal and proteasomal proteins.  our study establishes isotopic amine dimethylation as a versatile tool for quantitative proteomic analysis of ffpe specimens and underlines proteome alterations in ccrcc. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords dimethylationformalin-fixationparaffin-embedment clear cell renal cell carcinomaissue-copyright-statement© the author 2015 