Pathology Tissue-quantitative Mass Spectrometry Analysis to Profile Histone Post-translational Modification Patterns in Patient Samples
2015; Elsevier BV; Volume: 15; Issue: 3 Linguagem: Inglês
10.1074/mcp.m115.054510
ISSN1535-9484
AutoresRoberta Noberini, Andrea Uggetti, Giancarlo Pruneri, Saverio Minucci, Tiziana Bonaldi,
Tópico(s)RNA modifications and cancer
ResumoHistone 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. Proteomics. 2013; 10: 389-400Crossref PubMed Scopus (35) Google Scholar). Moreover, a few studies showed the possibility to globally analyze protein post-translational modifications, such as glycosylation and phosphorylation, from fixed and embedded tissues (10.Tian Y. Gurley K. Meany D.L. Kemp C.J. Zhang H. N-linked glycoproteomic analysis of formalin-fixed and paraffin-embedded tissues.J. Proteome Res. 2009; 8: 1657-1662Crossref PubMed Scopus (32) Google Scholar, 11.Ostasiewicz P. Zielinska D.F. Mann M. Wisniewski J.R. Proteome, phosphoproteome, and N-glycoproteome are quantitatively preserved in formalin-fixed paraffin-embedded tissue and analyzable by high-resolution mass spectrometry.J. Proteome Res. 2010; 9: 3688-3700Crossref PubMed Scopus (192) Google Scholar, 12.Wakabayashi M. Yoshihara H. Masuda T. Tsukahara M. Sugiyama N. Ishihama Y. Phosphoproteome analysis of formalin-fixed and paraffin-embedded tissue sections mounted on microscope slides.J. Proteome Res. 2014; 13: 915-924Crossref PubMed Scopus (39) Google Scholar). Here, we report for the first time the successful application of MS-based analysis of hPTMs to human clinical samples, focusing in particular on the development and validation of a method (PAT-H-MS) to extract histones from FFPE tissues in yield and purity sufficient to enable the subsequent use of a proteomic workflow optimized for hPTM analysis (13.Soldi M. Cuomo A. Bonaldi T. Improved bottom-up strategy to efficiently separate hypermodified histone peptides through ultra-HPLC separation on a bench top Orbitrap instrument.Proteomics. 2014; 14: 2212-2225Crossref PubMed Scopus (26) Google Scholar). By using this method we were able to profile in a quantitative manner 24 distinct modified histone peptides from human FFPE breast cancer samples belonging to different subtypes, identifying differences in histone methylation patterns of potential clinical relevance. Thus, PAT-H-MS represents a valid approach for hPTM analysis of clinical samples. Leukemic blasts were isolated from acute promyelocytic leukemia transgenic mice and 1 × 106 cells were i.v. injected in a syngenic recipient mouse to induce secondary leukemia (14.Minucci S. Monestiroli S. Giavara S. Ronzoni S. Marchesi F. Insinga A. Diverio D. Gasparini P. Capillo M. Colombo E. Matteucci C. Contegno F. Lo-Coco F. Scanziani E. Gobbi A. Pelicci P.G. PML-RAR induces promyelocytic leukemias with high efficiency following retroviral gene transfer into purified murine hematopoietic progenitors.Blood. 2002; 100: 2989-2995Crossref PubMed Scopus (94) Google Scholar). After massive splenomegaly was established, the mouse was sacrificed and its spleen was divided into two portions. One portion was washed and homogenized in 5 ml ice-cold phosphate buffered saline (PBS: 0.8% NaCl; 0.02% KCl; 0.02% KH2PO4; 0.2% Na2HPO4, pH 7.4) using a Dounce homogenizer, obtaining spleen cells that were counted, pelleted by centrifugation, rapidly frozen, and stored at −80 °C until use. The other half of the spleen was washed in PBS and incubated for 16 h at room temperature in a 4% paraformaldehyde solution. The fixed spleen was then routinely dehydrated with increasing concentrations of ethanol (70%, 80%, 90 and 100%) and subsequently included in paraffin using a tissue processor (Leica ASP300). Frozen cells and FFPE tissues were prepared similarly from mouse liver. Experimental procedures involving animals complied with the Guidelines of the Italian National Institute of Health, and were approved by the Institutional Ethical Committee. Invasive breast cancer specimens were obtained from 23 patients with duct invasive carcinoma not otherwise specified (supplemental Fig. S3A and supplemental Table S3), who were subjected to mastectomy or breast conserving surgery. The patients provided informed consent and this study was approved by the Ethical Committee of the European Institute of Oncology. Tumor samples were collected and snap frozen or fixed 4% formalin and embedded in paraffin. Tumor cells accounted for 50% or more of the samples, as evaluated histologically. The assessment of hormone receptors and Her-2 status, as well as the Ki-67 labeling index was ascertained by immunohistochemistry, according to the ASCO/CAP recommendations, using the FDA-approved anti-ER/PgR (PharmDX, Dako) and Her-2 (Herceptest, Dako), as well as the anti-Ki-67 antibody MIB-1. Breast cancer subtypes were defined according to the 2013 S. Gallen consensus conference recommendations, using immunohistochemical surrogates as follows: Luminal A-like: ER and/or PgR(+), HER2(-), Ki67 < 20%; Luminal B-like: ER and/or PgR(+), HER2(-), Ki67 ≥ 20; Triple Negative: ER, PgR and HER2(-), irrespective of Ki67 score; HER2-positive: HER2(+), irrespective of ER, PgR or Ki67. ER/PgR positivity was defined as ≥1% of immunoreactive neoplastic cells; HER2 positivity was defined as >10% of neoplastic cells with strong and continuous staining of the cell membrane (3+ by immunohistochemistry) and/or amplified by in situ hybridization techniques, in accordance to the ASCO/CAP guidelines. Four 10-μm tissue sections (corresponding to ∼20–60 mg of tissue, as measured by weighting the tissue after paraffin removal) were deparaffinized by adding 1 ml of hystolemon (Dasit Group Carlo Erba), vortexing for 30 s and centrifuging at 18,000 × g for 3 min (5 times). The samples were rehydrated with incubations in decreasing concentrations of ethanol (100%, 95%, 70%, 50%, 20%, and water) for 3 min at room temperature, followed by centrifugation for 3–5 min at 18,000 × g for each step. Rehydrated FFPE sections were permeabilized in 0.5 ml of Tris-buffered saline (TBS) containing 0.5% Tween20 and protease inhibitors for 20 min at room temperature in a rotating platform, followed by a 5 min centrifugation at 18,000 × g. The samples were then resuspended in 200 μl of 20 mm Tris pH 7.4 containing 2% SDS and sonicated in a Branson Digital Sonifier 250 with a 3 mm microtip until tissues pieces were homogenized. Proteins were extracted and de-crosslinked with a 45 min incubation at 95 °C, followed by a 4 h incubation at 65 °C. Protein concentration was estimated with the Bio-Rad DC protein assay kit (Bio-Rad, Segrate, Italy) and 16–20 μg of proteins were run on a 17% SDS-PAGE gel following protein detection with colloidal Coomassie staining (Expedeon, San Diego, CA) to quantify histone concentration based on a comparison with known amounts of recombinant histone H3.1 (New England Biolabs, Ipswich, MA). Acidic extraction, a step typically performed to purify histones, was omitted because it cannot be performed in the presence of detergents. Nevertheless, histones represented a good portion of the FFPE protein extract (supplemental Fig. S3B). The reproducibility of the histone isolation protocol was assessed by analyzing adjacent FFPE sections from the same mouse spleen (supplemental Fig. S2). Cells were obtained from fresh mouse spleen and liver as described above and histones were isolated as previously described (15.Cuomo A. Moretti S. Minucci S. Bonaldi T. SILAC-based proteomic analysis to dissect the “histone modification signature” of human breast cancer cells.Amino Acids. 2011; 41: 387-399Crossref PubMed Scopus (63) Google Scholar) (supplemental Fig. S1C). Briefly, 30 × 106 cells were resuspended in lysis buffer (10% sucrose; 0.5 mm EGTA, pH 8.0; 15 mm NaCl; 60 mm KCl; 15 mm HEPES; 0.5% Triton; 0.5 mm PMSF; 1 mm DTT; 5 mm NaF; 5 mm Na3VO4; 5 mm Na-butyrate; protease inhibitors) and nuclei were separated from the cytoplasm by centrifugation on a sucrose cushion. Histones were extracted by a 4 hours incubation in 0.4 N HCl at 4 °C and dialyzed overnight against 100 mm CH3COOH, using dialysis membranes with a 6–8 kDa cutoff (Spectrum Laboratories, Inc, Rancho Dominguez, CA). The dialyzed samples were lyophilized and stored at −80 °C. The same procedure was used to isolate histones from the breast cancer cell lines used to generate the super-SILAC mix (supplemental Fig.S1D, see below). Because breast tissue cannot be readily homogenized to single cells as described above for spleen and liver tissues and is available in limited amounts, we developed and alternative protocol to obtain histones. Twenty to seventy mg of frozen breast cancer tissue were thawed on ice, cut in pieces as small as possible with scissors and then homogenized in PBS containing 0.1% Triton X-100 and protease inhibitors using a Dounce homogenizer. Tissue debris were removed by filtering the homogenate through a 100 μm cell strainer and nuclei were isolated with a 10 min centrifugation at 2300 × g. Nuclei were resuspended in 100–200 μl of the same buffer containing 0.1% SDS and incubated for few minutes at 37 °C in the presence of 250 U of benzonase (Merk Millipore, Darmstadt, Germany) to digest nucleic acids. No acidic extraction was performed to avoid samples loss. The concentration of purified histones and nuclei extracts was measured using the Bradford protein assay kit (Thermo Fisher Scientific) and their purity was assessed by SDS-PAGE. Yield ranged between 0.5 and 1.3 μg of histone octamer per 10 mg of starting tissue. About 5 μg of histones per run per sample were separated on a 17% SDS-PAGE gel and bands corresponding to histones H3 and H4 were excised and in-gel digested as previously described (15.Cuomo A. Moretti S. Minucci S. Bonaldi T. SILAC-based proteomic analysis to dissect the “histone modification signature” of human breast cancer cells.Amino Acids. 2011; 41: 387-399Crossref PubMed Scopus (63) Google Scholar). Briefly, gel bands were cut in pieces and destained with repeated washes in 50% acetonitrile (ACN) in H2O, alternated with dehydration steps in 100% ACN. Gel pieces were then in-gel chemically alkylated with D6-acetic anhydride (Sigma-Aldrich) 1:9 in 1 m NH4HCO3, using CH3COONa as catalyzer. After shaking for 3 h at 37 °C, chemically modified gel slices were washed with NH4HCO3, alternated with ACN at increasing percentages (from 50 to 100). The in-gel digestion was performed overnight with 100 ng/μl trypsin (Promega) in 50 mm NH4HCO3 at 37 °C, in order to obtain an “Arg-C like” in-gel digestion that originates histone peptides of optimal length for MS analysis by cleaving at the C-terminal of arginine residues. Finally, digested peptides were extracted using 5% formic acid alternated with ACN 100%. In SILAC experimental set-ups, unlabeled and heavy-labeled histones were mixed in equal amounts prior to gel separation, and then processed as described above. Digested peptides were desalted and concentrated using a combination of reversed-phase C18/C and strong cation exchange (SCX) chromatography on handmade nanocolumns (StageTips). Digested peptides were then eluted with 80% ACN/0.5% acetic acid and 5% NH4OH/30% methanol from C18/C and SCX StageTips, respectively. Eluted peptides were lyophilized, resuspended in 1% TFA, pooled and subjected to LC-MS/MS analysis. Peptide mixtures were separated by reversed-phase chromatography on an in-house-made 25 cm column (inner diameter 75 μm, outer diameter 350 μm outer diameter, 1.9 μm ReproSil, Pur C18AQ medium), using a ultra nanoflow high-performance liquid chromatography (HPLC) system (EASY-nLC™ 1000, Thermo Fisher Scientic) connected online to a Q Exactive instrument (Thermo Fisher Scientific) through a nanoelectrospray ion source. Solvent A was 0.1% formic acid (FA) in ddH2O and solvent B was 80% ACN plus 0.1% FA. Peptides were injected in an aqueous 1% TFA solution at a flow rate of 500 nl/min and were separated with a 100 min linear gradient of 0–40% solvent B, followed by a 5 min gradient of 40–60% and a 5 min gradient of 60–95% at a flow rate of 250 nl/min. The Q Exactive instrument was operated in the data-dependent acquisition (DDA) mode to automatically switch between full scan MS and MS/MS acquisition. Survey full scan MS spectra (m/z 300–1650) were analyzed in the Orbitrap detector with resolution of 35,000 at m/z 400. The five most intense peptide ions with charge states ≥ 2 were sequentially isolated to a target value for MS1 of 3 × 106 and fragmented by HCD with a normalized collision energy setting of 25%. The maximum allowed ion accumulation times were 20 msec for full scans and 50 msec for MS/MS and the target value for MS/MS was set to 1 × 106. The dynamic exclusion time was set to 20 s and the standard mass spectrometric conditions for all experiments were as follows: spray voltage of 2.4 kV, no sheath and auxiliary gas flow. One technical replicate of the breast cancer cell lines profiled to set up the super-SILAC strategy (see below) was analyzed through HPLC in combination with a LTQ-Velos Orbitrap. Samples were separated by nano-liquid chromatography using an EASY-nLC system (Proxeon Biosystems, Odense, Denmark) on an in-house-made 50 cm column (inner diameter 75 μm, outer diameter 350 μm outer diameter, 3 μm ReproSil, Pur C18AQ medium) and analyzed using an LTQ-Velos Orbitrap instrument (Thermo Fisher Scientific). The solvent composition were as described above, and peptides were separated with a shallow gradient at a flow rate of 300 nl/min: 10–50% solvent B over 95 min, 50–60% over 5 min and 60–80% over 5 min. The LTQ-Velos Orbitrap was operated in DDA mode and MS spectra (m/z range 300–1650) were analyzed with resolution r = 30,000 at m/z 400 (transient time = 250 ms). The 10 most intense peptide ions with charge states ≥ 2 were sequentially isolated to a target value of 1 × 106 and fragmented by CID with a normalized collision energy setting of 35%. MS/MS settings were: maximum ion injection time 150 ms, minimum signal threshold 500; isolation width 2 Da; ion target value 1e4, dynamic exclusion time 25 s. Results obtained using the two LC/MS setups generated very similar results (supplemental Fig. S4C). Acquired RAW data were analyzed by the integrated MaxQuant software v.1.3.0.5, which performed peak list generation and protein identification using the Andromeda search engine (16.Cox J. Neuhauser N. Michalski A. Scheltema R.A. Olsen J.V. Mann M. Andromeda: a peptide search engine integrated into the MaxQuant environment.J. Proteome Res. 2011; 10: 1794-1805Crossref PubMed Scopus (3448) Google Scholar). The Uniprot MOUSE 1301 (33202 entries) and HUMAN 1301 (43990 entries) databases were used for peptide identification. Enzyme specificity was set to Arg-C. The estimated false discovery rate of all peptide identifications was set at a maximum of 1%. The mass tolerance was set to 6 ppm for precursor and fragment ions. Three missed cleavages were allowed, and the minimum peptide length was set to 6 amino acids. We focused on lysine methylation and acetylation, including as variable modifications lysine D3-acetylation (+45.0294 Da), lysine monomethylation (+ 59.0454, corresponding to the sum of D3-acetylation (+45.0294) and monomethylation (+14.016 Da)), dimethylation (+28.031 Da), trimethylation (+42.046 Da), and lysine acetylation (+42.010 Da). To reduce the search time and the rate of false positives, which increase with increasing the number of variable modifications included in the database search (17.Ong S.E. Mittler G. Mann M. Identifying and quantifying in vivo methylation sites by heavy methyl SILAC.Nat. Methods. 2004; 1: 119-126Crossref PubMed Scopus (365) Google Scholar), the raw data were analyzed through multiple parallel MaxQuant jobs (18.Bremang M. Cuomo A. Agresta A.M. Stugiewicz M. Spadotto V. Bonaldi T. Mass spectrometry-based identification and characterisation of lysine and arginine methylation in the human proteome.Mol. Biosyst. 2013; 9: 2231-2247Crossref PubMed Scopus (116) Google Scholar), setting different combinations of variable modifications: (1.Jenuwein T. Allis C.D. Translating the histone code.Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7632) Google Scholar) D3-acetylation, lysine monomethylation with D3-acetylation, dimethylation and lysine acetylation, (2.Portela A. Esteller M. Epigenetic modifications and human disease.Nat. Biotechnol. 2010; 28: 1057-1068Crossref PubMed Scopus (1988) Google Scholar) D3-acetylation, lysine monomethylation with D3-acetylation, dimethylation and trimethylation, (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) D3-acetylation, lysine monomethylation with D3-acetylation, trimethylation, and lysine acetylation. In addition, for the mouse samples we included in the search lysine modifications that may be induced by formalin fixation (19.Zhang Y. Muller M. Xu B. Yoshida Y. Horlacher O. Nikitin F. Garessus S. Magdeldin S. Kinoshita N. Fujinaka H. Yaoita E. Hasegawa M. Lisacek F. Yamamoto T. Unrestricted modification search reveals lysine methylation as major modification induced by tissue formalin fixation and paraffin embedding.Proteomics. 2015; 15: 2568-2579Crossref PubMed Scopus (34) Google Scholar): formylation (+27.9949 Da), methylene adducts (+12 Da) and methylol adducts (+30.0106 Da), which were included in a fourth search job together with D3-acetylation. MaxQuant search results from different jobs were exported and combined; peptides with Andromeda score less than 60 (corresponding to a Mascot score of 15 (16.Cox J. Neuhauser N. Michalski A. Scheltema R.A. Olsen J.V. Mann M. Andromeda: a peptide search engine integrated into the MaxQuant environment.J. Proteome Res. 2011; 10: 1794-1805Crossref PubMed Scopus (3448) Google Scholar), which has been previously used as a cut-off value (20.Jung H.R. Pasini D. Helin K. Jensen O.N. Quantitative mass spectrometry of histones H3.2 and H3.3 in Suz12-deficient mouse embryonic stem cells reveals distinct, dynamic post-translational modifications at Lys-27 and Lys-36.Mol. Cell. Proteomics. 2010; 9: 838-850Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar)) and localization probability score less than 0.75 (21.Pan C. Gnad F. Olsen J.V. Mann M. Quantitative phosphoproteome analysis of a mouse liver cell line reveals specificity of phosphatase inhibitors.Proteomics. 2008; 8: 4534-4546Crossref PubMed Scopus (87) Google Scholar, 22.Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2807) Google Scholar), were removed. Modified peptide parameters and representative MS/MS spectra for each modified peptide are reported in supplemental Table S4 and supplemental Fig. S8, respectively. Identifications and retention times were used to guide the manual quantification of each modified peptide using QualBrowser version 2.0.7 (ThermoFisher Scientific, Waltham, MA). Site assignment was evaluated using QualBrowser and MaxQuant Viewer 1.3.0.5. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (23.Vizcaino J.A. Deutsch E.W. Wang R. Csordas A. Reisinger F. Rios D. Dianes J.A. Sun Z. Farrah T. Bandeira N. Binz P.A. Xenarios I. Eisenacher M. Mayer G. Gatto L. Campos A. Chalkley R.J. Kraus H.J. Albar J.P. Martinez-Bartolome S. Apweiler R. Omenn G.S. Martens L. Jones A.R. Hermjakob H. ProteomeXchange provides globally coordinated proteomics data submission and dissemination.Nat. Biotechnol. 2014; 32: 223-226Crossref PubMed Scopus (2070) Google Scholar) via the PRIDE partner repository with the data set identifier PXD002669. Extracted ion chromatograms (XIC) were constructed for each doubly charged precursor based on its m/z value, using a mass tolerance of 10 ppm and a mass precision up to four decimals. For each histone modified peptide, the relative abundance (RA) was estimated by dividing the area under the curve (AUC) of each modified peptide for the sum of the areas corresponding to all the observed forms of that peptide (24.Pesavento J.J. Mizzen C.A. Kelleher N.L. Quantitative analysis of modified proteins and their positional isomers by tandem mass spectrometry: human histone H4.Anal. Chem. 2006; 78: 4271-4280Crossref PubMed Scopus (201) Google Scholar). For SILAC experiments, Arg10 was selected as heavy label (multiplicity = 2) in MaxQuant. The heavy form of each modified peptide was quantified from its XIC and the relative abundance quantified. L/H ratios of relative abundances were calculated for each modified peptide (supplemental Fig. S5A). Because we assume that the sum of all the possible forms of the peptide should be constant in all the samples, using ratios of relative abundances corrects for possible errors in mixing light and heavy histones. To better visualize differences among biopsies the ratio of one sample relative to the standard was divided by the average ratios across the samples (supplemental Fig. S5C). As an alternative normalization strategy, SILAC ratios were obtained from light and heavy AUC values and were corrected based on the SILAC ratio of an unmodified histone H3 peptides (supplemental Fig. S5B and S5D). The results obtained using the two normalization strategies were very similar, suggesting that other possible modifications not specifically indicated in the database search do not substantially affect the quantification based on the estimation of %RA. Visualization of hPTM ratios and unsupervised hierarchical clustering were performed using Perseus, setting correlation distance and complete linkage as parameters (http://www.perseus-framework.org/). FFPE and frozen samples from mouse tissues were acquired
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