Pathology Tissue-quantitative Mass Spectrometry Analysis to Profile Histone Post-translational Modification Patterns in Patient Samples

Abstract

Histone post-translational modifications (hPTMs) generate a complex combinatorial code that has been implicated with various pathologies, including cancer. Dissecting such a code in physiological and diseased states may be exploited for epigenetic biomarker discovery, but hPTM analysis in clinical samples has been hindered by technical limitations. Here, we developed a method (PAThology tissue analysis of Histones by Mass Spectrometry - PAT-H-MS) that allows to perform a comprehensive, unbiased and quantitative MS-analysis of hPTM patterns on formalin-fixed paraffin-embedded (FFPE) samples. In pairwise comparisons, histone extracted from formalin-fixed paraffin-embedded tissues showed patterns similar to fresh frozen samples for 24 differentially modified peptides from histone H3. In addition, when coupled with a histone-focused version of the super-SILAC approach, this method allows the accurate quantification of modification changes among breast cancer patient samples. As an initial application of the PAThology tissue analysis of Histones by Mass Spectrometry method, we analyzed breast cancer samples, revealing significant changes in histone H3 methylation patterns among Luminal A-like and Triple Negative disease subtypes. These results pave the way for retrospective epigenetic studies that combine the power of MS-based hPTM analysis with the extensive clinical information associated with formalin-fixed paraffin-embedded archives. Histone post-translational modifications (hPTMs) generate a complex combinatorial code that has been implicated with various pathologies, including cancer. Dissecting such a code in physiological and diseased states may be exploited for epigenetic biomarker discovery, but hPTM analysis in clinical samples has been hindered by technical limitations. Here, we developed a method (PAThology tissue analysis of Histones by Mass Spectrometry - PAT-H-MS) that allows to perform a comprehensive, unbiased and quantitative MS-analysis of hPTM patterns on formalin-fixed paraffin-embedded (FFPE) samples. In pairwise comparisons, histone extracted from formalin-fixed paraffin-embedded tissues showed patterns similar to fresh frozen samples for 24 differentially modified peptides from histone H3. In addition, when coupled with a histone-focused version of the super-SILAC approach, this method allows the accurate quantification of modification changes among breast cancer patient samples. As an initial application of the PAThology tissue analysis of Histones by Mass Spectrometry method, we analyzed breast cancer samples, revealing significant changes in histone H3 methylation patterns among Luminal A-like and Triple Negative disease subtypes. These results pave the way for retrospective epigenetic studies that combine the power of MS-based hPTM analysis with the extensive clinical information associated with formalin-fixed paraffin-embedded archives. Histone post-translational modifications (hPTMs) 1The abbreviations used are:hPTMhistone post-translational modificationsPAT-H-MS FFPEpathology tissue analysis of histones by mass spectrometryFFPEformalin-fixed paraffin embeddedMS/MStandem mass spectrometryRArelative abundanceXICExtracted ion chromatogramsSDS-PAGEsodium dodecyl sulphate-polyacrylamide gel electrophoresisgeLC-MSSDS-PAGE followed by in-gel digestion and peptide analysis by mass spectrometrySILACstable isotope labeling by amino acid in cell cultureArg0unlabeled L-arginineArg1013C6 15N4 L-arginineLys0unlabeled L-lysineLys813C6 15N2 L-lysineFDRfalse discovery rateHheavy-labeledLlight-labeled. generate a complex combinatorial code that plays a critical role during the physiological and pathological regulation of gene expression (1.Jenuwein T. Allis C.D. Translating the histone code.Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7632) Google Scholar). Alterations in histone modification patterns have been linked with various diseases, including cancer, often as a result of the aberrant expression or localization of histone modifying enzymes (2.Portela A. Esteller M. Epigenetic modifications and human disease.Nat. Biotechnol. 2010; 28: 1057-1068Crossref PubMed Scopus (1988) Google Scholar). Therefore, accurately dissecting hPTM patterns in normal and diseased tissues could yield epigenetic biomarkers useful for prognostic, diagnostic, and therapeutic purposes. Immunohistochemistry studies have shown the potential of this strategy (3.Seligson D.B. Horvath S. Shi T. Yu H. Tze S. Grunstein M. Kurdistani S.K. Global histone modification patterns predict risk of prostate cancer recurrence.Nature. 2005; 435: 1262-1266Crossref PubMed Scopus (880) Google Scholar, 4.Seligson D.B. Horvath S. McBrian M.A. Mah V. Yu H. Tze S. Wang Q. Chia D. Goodglick L. Kurdistani S.K. Global levels of histone modifications predict prognosis in different cancers.Am. J. Pathol. 2009; 174: 1619-1628Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar), but they were limited to the analysis of only a few hPTMs. In addition, despite their sensitivity and ease of use, antibody-based assays are hindered by issues such as the difficulty in detecting adjacent modifications and the limited linearity of the signal. As an alternative to traditional antibody-based methods, in recent years MS has become the elective method to analyze hPTMs, thanks to its unbiased nature, accuracy and its ability to quantitate modifications and detect their combinations. Various MS-based workflows optimized for hPTM analysis have been developed (5.Soldi M. Cuomo A. Bremang M. Bonaldi T. Mass spectrometry-based proteomics for the analysis of chromatin structure and dynamics.Int. J. Mol. Sci. 2013; 14: 5402-5431Crossref PubMed Scopus (25) Google Scholar), but most of the studies focused on cell lines and animal tissue, whereas the potential offered by the analysis of clinical samples has been left largely unexploited. In particular, the MS-based analysis of hPTMs from formalin-fixed paraffin-embedded (FFPE) samples has never been addressed. histone post-translational modifications pathology tissue analysis of histones by mass spectrometry formalin-fixed paraffin embedded tandem mass spectrometry relative abundance Extracted ion chromatograms sodium dodecyl sulphate-polyacrylamide gel electrophoresis SDS-PAGE followed by in-gel digestion and peptide analysis by mass spectrometry stable isotope labeling by amino acid in cell culture unlabeled L-arginine 13C6 15N4 L-arginine unlabeled L-lysine 13C6 15N2 L-lysine false discovery rate heavy-labeled light-labeled. Paraffin embedding following fixation in buffered formalin is the storage method of choice for clinical specimens, thus representing an invaluable source of clinical samples linked to retrospective patient information. Large formalin-fixed paraffin embedded (FFPE) archives, which are available in many hospitals, have been successfully exploited for DNA and RNA analyses, including chromatin immunoprecipitation (6.Amatori S. Ballarini M. Faversani A. Belloni E. Fusar F. Bosari S. Pelicci P.G. Minucci S. Fanelli M. PAT-ChIP coupled with laser microdissection allows the study of chromatin in selected cell populations from paraffin-embedded patient samples.Epigenetics Chromatin. 2014; 7: 18Crossref PubMed Scopus (17) Google Scholar, 7.Fanelli M. Amatori S. Barozzi I. Soncini M. Dal Zuffo R. Bucci G. Capra M. Quarto M. Dellino G.I. Mercurio C. Alcalay M. Viale G. Pelicci P.G. Minucci S. Pathology tissue-chromatin immunoprecipitation, coupled with high-throughput sequencing, allows the epigenetic profiling of patient samples.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 21535-21540Crossref PubMed Scopus (52) Google Scholar). However, the extensive protein cross-linking generated by formaldehyde fixation has hindered the proteomic study of this type of tissue. This problem has been addressed and overcome only recently in global proteomic studies by taking advantage of extraction protocols based on heat-induced antigen retrieval techniques derived from immunohistochemistry (8.Palmer-Toy D.E. Krastins B. Sarracino D.A. Nadol Jr., J.B. Merchant S.N. Efficient method for the proteomic analysis of fixed and embedded tissues.J. Proteome Res. 2005; 4: 2404-2411Crossref PubMed Scopus (112) Google Scholar, 9.Fowler C.B. O'Leary T.J. Mason J.T. Toward improving the proteomic analysis of formalin-fixed, paraffin-embedded tissue.Expert Rev. 2013; PubMed Scopus Google Scholar). a few studies showed the to analyze protein post-translational such as and from fixed and embedded tissues H. analysis of formalin-fixed and paraffin-embedded tissues.J. Proteome Res. 2009; PubMed Scopus Google Scholar, M. and are in formalin-fixed paraffin-embedded tissue and by mass Proteome Res. 2010; PubMed Scopus Google Scholar, M. H. T. M. analysis of formalin-fixed and paraffin-embedded tissue on Proteome Res. 2014; PubMed Scopus Google Scholar). Here, we for the the application of MS-based analysis of hPTMs to human clinical samples, in on the and of a method to histones from tissues in yield and to the of a proteomic optimized for hPTM analysis M. Cuomo A. Bonaldi T. strategy to histone peptides on a 2014; 14: PubMed Scopus Google Scholar). this method we were to in a quantitative 24 modified histone peptides from human breast cancer samples to different in histone methylation patterns of potential clinical a for hPTM analysis of clinical samples. were from and were in a to S. S. S. S. F. A. D. M. E. C. F. F. E. A. Pelicci P.G. with following gene PubMed Scopus Google Scholar). the and its and in buffered a that were by and of the in and for in a fixed with of and and in paraffin a tissue and tissues were from with the of the of and were by the breast cancer were from with and were to or breast and this study by the of the of samples were and frozen or fixed formalin and embedded in for or of the samples, as of and as as the labeling by to the the and as as the cancer were to the S. as Luminal Luminal Triple and of of or as of as of with and of the cell by by in in to the tissue to of tissue, as by the tissue paraffin were by of for and for samples were with in of and for followed by for for were in of and for in a followed by a samples were in of and in a with a tissues were were extracted and with a followed by a with the protein and of were on a SDS-PAGE gel following protein with to histone based on a with of histone a to be in the of histones a of the protein of the histone by adjacent from the were from fresh and as and histones were as A. S. Minucci S. Bonaldi T. proteomic analysis to the modification of human breast cancer PubMed Scopus Google were in and were from the by on a Histones were extracted by a in and with a samples were and used to histones from the breast cancer cell lines used to generate the super-SILAC breast tissue be to as for and tissues and is available in limited we developed and alternative to to of frozen breast cancer tissue were on in as as with and in and a were by the a cell and were with a were in of the and for few in the of of to extraction to samples of histones and the protein and their by and of histone of tissue. of histones were on a SDS-PAGE gel and to histones H3 and were and in-gel as A. S. Minucci S. Bonaldi T. proteomic analysis to the modification of human breast cancer PubMed Scopus Google Scholar). gel were in and with in in with in were in-gel with in as for modified gel were with with to in-gel digestion with in in to an in-gel digestion that histone peptides of for MS analysis by the of peptides were extracted acid with In unlabeled and heavy-labeled histones were in to gel and as peptides were and a of and on peptides were with acid and from and peptides were in and to were by on an a to a a ion acid in and were in an a rate of and were with a of followed by a of and a of a rate of in the to MS and MS were analyzed in the with of most peptide with states were to a for of and by with a of ion were for and for and the for to to and the mass for were as of and technical of the breast cancer cell lines to the super-SILAC strategy analyzed in with a were by an on an and analyzed an were as and peptides were with a a rate of and in and MS were analyzed with most peptide with states were to a of and by with a of ion ion the generated similar results were analyzed by the which and protein the J. A. M. a peptide the Proteome Res. PubMed Scopus Google Scholar). and were used for peptide to false discovery rate of peptide a of mass to for and were and the peptide to amino focused on methylation and including as modifications to the of and and the and the rate of false which with the of modifications in the G. M. and in methylation by PubMed Scopus Google Scholar), the were analyzed M. Cuomo A. M. V. Bonaldi T. Mass spectrometry-based and of and methylation in the human 2013; PubMed Scopus Google Scholar), different of (1.Jenuwein T. Allis C.D. Translating the histone code.Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7632) Google with and (2.Portela A. Esteller M. Epigenetic modifications and human disease.Nat. Biotechnol. 2010; 28: 1057-1068Crossref PubMed Scopus (1988) Google with and (3.Seligson D.B. Horvath S. Shi T. Yu H. Tze S. Grunstein M. Kurdistani S.K. Global histone modification patterns predict risk of prostate cancer recurrence.Nature. 2005; 435: 1262-1266Crossref PubMed Scopus (880) Google with and In addition, for the samples we in the modifications that may be by formalin fixation M. B. F. S. S. H. E. M. F. T. modification methylation as modification by tissue formalin fixation and paraffin PubMed Scopus Google and which were in a with results from different were and peptides with to a of J. A. M. a peptide the Proteome Res. PubMed Scopus Google Scholar), which has been used as a D. mass spectrometry of histones and in post-translational modifications and 2010; Full Text Full Text PDF PubMed Scopus Google and localization C. F. M. analysis of a cell of PubMed Scopus Google Scholar, B. F. B. C. M. in and in Full Text Full Text PDF PubMed Scopus Google Scholar), were peptide and for modified peptide are in and and were used to the quantification of modified peptide version and mass spectrometry proteomics have been to the Wang R. A. F. D. T. I. M. G. L. A. S. R. L. H. proteomics and Biotechnol. 2014; PubMed Scopus Google the with the Extracted ion chromatograms were for based on its a mass of and a mass to histone modified the relative abundance by the the of modified peptide for the of the to the of that peptide analysis of modified and their by tandem mass human histone PubMed Scopus Google Scholar). selected as in of modified peptide from its and the relative abundance of relative were for modified peptide we that the of the of the peptide be in the samples, of relative for in and among the of relative to the by the the samples As an alternative were from and and were based on the of an histone H3 peptides and results the were that modifications in the the quantification based on the of of hPTM and were and as and frozen samples from tissues were in for samples with the of samples, for which were and frozen samples from human breast cancer were in analysis of patient for among breast cancer among for breast cancer were analyzed by followed by Luminal A-like and Triple Negative samples were by cancer and cell lines were from were with human human and human cancer were in with of and cancer were for in to a of cell lines that the of modifications in breast samples, we analyzed the hPTM patterns of breast cancer cell lines to different and with different this we used a A. S. Minucci S. Bonaldi T. proteomic analysis to the modification of human breast cancer PubMed Scopus Google Scholar), unlabeled normal breast are of the heavy-labeled breast cancer cell samples and used as an for histone H3 hPTM patterns of and of based on of relative they showed the most we and to generate a super-SILAC that be breast cancer samples to analysis were used for coupled with a or a with a which the results the of the results different of histones from and were and or frozen were by on and by with and H3 used as were on a and the the to by mass spectrometry hPTMs from tissue we used different which be in and the to and frozen tissue from the samples for to analyzed samples from or the of the to different and that were in paraffin for or an to the of storage that the hPTMs in the samples are of the epigenetic of the tissue, for we used as histones a from frozen derived from the tissue and for the of were extracted from samples a that on the of of and the that have been shown to be critical to from tissues and to the generated by formaldehyde fixation C.B. O'Leary T.J. Mason J.T. Toward improving the proteomic analysis of formalin-fixed, paraffin-embedded tissue.Expert Rev. 2013; PubMed Scopus Google Scholar). In particular, of have been shown to be the most way to the of from tissues and their Histones be from paraffin few (1.Jenuwein T. Allis C.D. Translating the histone code.Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7632) Google and (2.Portela A. Esteller M. Epigenetic modifications and human disease.Nat. Biotechnol. 2010; 28: 1057-1068Crossref PubMed Scopus (1988) Google and by in a (3.Seligson D.B. Horvath S. Shi T. Yu H. Tze S. Grunstein M. Kurdistani S.K. Global histone modification patterns predict risk of prostate cancer recurrence.Nature. 2005; 435: 1262-1266Crossref PubMed Scopus (880) Google protein extraction and and D.B. Horvath S. McBrian M.A. Mah V. Yu H. Tze S. Wang Q. Chia D. Goodglick L. Kurdistani S.K. Global levels of histone modifications predict prognosis in different cancers.Am. J. Pathol. 2009; 174: 1619-1628Abstract Full Text Full Text PDF PubMed Scopus (406) Google which and and histones from in the are as histones a of the based on protein on the with of histones on a SDS-PAGE extraction from paraffin of of histone Histone yield among different tissues from samples but for the of tissues the extraction for a MS-based of hPTMs, which of histone MS gel to histones H3 and and in-gel a that with followed by digestion T. J.T. A. of mass spectrometry for the analysis of histone PubMed Scopus Google Scholar). This allows to from gel an digestion that histone peptides of for the following analysis by which coupled to a the MS in the we the most various from formalin in to and methylation the were for and modifications by formalin modifications in frozen samples are in we the of on histone H3 and and on the histone and few on peptide H3 and peptide which were in the frozen samples. the levels of modifications we the abundance of modified peptide ion chromatograms and to relative abundance by the the of modified peptide for the of the to the for that peptide analysis of modified and their by tandem mass human histone PubMed Scopus Google of the modified peptides only in samples, such a that they could be have a relative abundance and thus representing a of the the and frozen tissues showed relative for the most and histone modifications in peptides from histone H3 and the and frozen tissues for peptides to the histone from adjacent from the that the frozen and is similar to the technical in samples. of samples storage showed to frozen tissues for the an in and abundance and the of on histone that for of the and These results are by the storage and are in with a recent analysis that methylation is the most modification by M. B. F. S. S. H. E. M. F. T. modification methylation as modification by tissue formalin fixation and paraffin PubMed Scopus Google Scholar). Therefore, we peptides H3 and from following on histone H3 peptides post-translational modifications and which are by to this to the hPTM analysis of samples cancer with a super-SILAC to be to accurately modification changes among patient samples. labeling by amino in cell culture strategy on labeling of cell populations of different amino which be by MS B. I. D.B. H. A. M. isotope labeling by amino in cell as a and accurate to expression Full Text Full Text PDF PubMed Scopus Google Scholar). the differentially samples be during the and are analyzed by the of during is and the accuracy of the of this the super-SILAC a of is unlabeled samples and used as an which allows and the of to the analysis of clinical samples T. J. M. for quantitative proteomics of human 2010; 7: PubMed Scopus Google Scholar). In addition, a of cell lines of a the advantage of representing the in a complex such as a tissue. super-SILAC has been successfully used only for global proteomic but to analyze the M. M. quantification in tissues by a PubMed Scopus Google Scholar), the T. M. of the by super-SILAC during breast cancer and in human 2013; Full Text Full Text PDF PubMed Scopus Google and histones Wang J. R. M. F. Wang E. L. J. J. Yu R. V. R. J. G. J. C. F. Global chromatin profiling in 2013; PubMed Scopus Google Scholar). Here, we used a histone-focused version of the super-SILAC to analyze and hPTMs in breast cancer samples. on their different of and hPTM patterns we breast cancer cell lines to generate a super-SILAC that breast cancer samples to analysis and used as an the to the analysis of breast samples from and to their fresh frozen tissues as for the of the samples, the SDS-PAGE gel used to the and the of the histones from frozen breast cancer tissue by a extraction that histones of the based on protein in for MS analysis from of frozen the results with samples, the quantification of the and the frozen tissues showed similar results for histone H3 peptides and we the and the super-SILAC of this type of is by that are a of the abundance of the peptides in breast cancer with the super-SILAC were by the to among samples and and hPTM changes among were similar for the of samples that were frozen or in paraffin of the were in frozen and samples, a few which may be to the that tissue were frozen or the of tissues an for the analysis of and tissue, which could be overcome by the with such as of or laser to techniques have been successfully used for the of pathology tissue chromatin immunoprecipitation an for the extraction and high-throughput analysis of chromatin that the of proteomic analysis (6.Amatori S. Ballarini M. Faversani A. Belloni E. Fusar F. Bosari S. Pelicci P.G. Minucci S. Fanelli M. PAT-ChIP coupled with laser microdissection allows the study of chromatin in selected cell populations from paraffin-embedded patient samples.Epigenetics Chromatin. 2014; 7: 18Crossref PubMed Scopus (17) Google Scholar, 7.Fanelli M. Amatori S. Barozzi I. Soncini M. Dal Zuffo R. Bucci G. Capra M. Quarto M. Dellino G.I. Mercurio C. Alcalay M. Viale G. Pelicci P.G. Minucci S. Pathology tissue-chromatin immunoprecipitation, coupled with high-throughput sequencing, allows the epigenetic profiling of patient samples.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 21535-21540Crossref PubMed Scopus (52) Google Scholar). cancer be Luminal A-like and Luminal and and Triple breast cancer hPTM patterns and a patterns and clinical 24 differentially modified peptides from histone H3 in samples from for clinical to the various by different levels of histone Luminal A-like samples, levels of histone H3 and and levels of and and Luminal A-like has been in immunohistochemistry studies D. S. S. J. M. L. C. A. M. Global and abundance in breast PubMed Scopus Google Scholar, M.A. R. A. of and histone with breast cancer in the Res. 2014; PubMed Scopus Google Scholar), thus an for which of of the Triple Negative samples and samples, for and Luminal samples, samples and Triple to levels of and but in levels of peptides on or and whereas has levels of and with the with levels of analysis a in hPTM levels in among Luminal A-like and Triple Negative breast cancer samples and analyzed the to and by analysis the levels of in Triple Negative samples a different similar by analysis of the samples and in frozen samples to the significant were in of antibody-based assays for the for which showed quantitative of and that the MS-based method developed may be in detecting among samples with traditional antibody-based that are by and to detect among samples, and that the in this by a but among samples. enzymes to chromatin by modifying histones are in clinical use, and are a clinical of of the epigenetic in is of to the of this to the and have to be to patient samples. results for the the to perform a comprehensive, unbiased and quantitative MS-analysis of histone patterns on patient samples for pathology which been to antibody-based with fresh that most of the analyzed histone H3 and are and in tissues to years with the of histone H3 peptide in samples for However, the of modifications a of the and the relative quantification of the most modifications on histone H3 peptides and on which we focused Therefore, extraction allows the and quantification of 24 histone peptide storage of samples. changes may be years of storage in paraffin in a few of the 24 modified histone that we focused the of among samples may from samples from the is with is available from most its in clinical with the traditional antibody-based in the for hPTM of modifications be in a MS including modifications for which This the of to a of hPTM hPTMs be accurately when such as a super-SILAC are used as an and allows to analyze samples of a when a clinical detect of which may modification of pathological states with power as with As a we used the in with a super-SILAC to hPTM changes in a clinical of human breast cancer to different subtypes. In with studies D. S. S. J. M. L. C. A. M. Global and abundance in breast PubMed Scopus Google Scholar, M.A. R. A. of and histone with breast cancer in the Res. 2014; PubMed Scopus Google Scholar), we that histone H3 or in with in Luminal A-like samples with and in samples to which Triple Negative and samples. This result the of that be used to information from patient samples. of the Triple Negative samples were in In to a in this showed an of or in with the samples to have the levels of expression among the samples the hPTM in this with with this the only Triple Negative that a different has a expression the samples to the breast a of for which have been and the of this of to that of of the Triple Negative samples that we analyzed were in their hPTM and in showed levels of the and levels of the and analysis of a of breast cancer is and the histone in this study Triple Negative samples from breast cancer subtypes. In addition, levels of histone are often associated with of histone modifying may epigenetic for the of Triple Negative the levels of and that we may be linked to the of of the which of J. the histone the of and PubMed Scopus Google Scholar). used in this could be in to epigenetic linked with patient cancer or clinical in breast cancer as as epigenetic a In by the of MS-based hPTM analysis with the clinical information associated with samples, a significant in the of clinical thus that to epigenetic and clinical by the of epigenetic biomarkers useful for the of cancer and A. A. and C. for technical I. and F. for the samples, L. and M. Ballarini for and A. for the breast cancer with