Collection of specimens and preparation of FFPE tissues
Spleen tissue was collected from a leukemic mouse with splenomegaly, 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).
Glioblastoma (GBM) specimens were collected from patients at the Department of Neurosurgery at Istituto Neurologico Carlo Besta. Human GBM-derived neurospheres were obtained and grown as previously described . Prior to injection in mice, the neurospheres were mechanically dissociated, and the cells were resuspended in 2 μL of phosphate-buffered saline and stereotaxically injected into the nucleus caudatus (1 mm posterior, 3 mm left lateral, 3.5 mm in depth from bregma) of 5-week-old female nu/nu CD1 mice (Charles River, Wilmington, MA). Whole mouse brains were collected and processed as described above.
Breast cancer frozen and FFPE specimens were obtained from patients with duct invasive carcinoma at the European Institute of Oncology, who were subjected to mastectomy or breast conserving surgery. FFPE resection specimens of duct invasive breast carcinoma for LMD were selected from the archive of the Institute of Pathology Heidelberg with the support of the National Center for Tumor Diseases (NCT, Heidelberg, Germany). Tumor cellularity was evaluated histologically, and the assessment of hormone and Her-2 receptors and the Ki-67 labeling index was performed as previously described . Luminal A-like and triple negative subtypes were defined as follows: luminal A-like: ER and/or PgR(+), HER2(−), Ki67 < 20%; triple negative: ER, PgR, and HER2(−), irrespective of Ki67 score.
Histones were isolated from FFPE tissues as recently described . Briefly, four 10-μm tissue sections were deparaffinized with four washes in hystolemon (Dasit Group Carlo Erba) and rehydrated from 100% ethanol to water (through intermediate incubations in ethanol 95, 70, 50, and 20%). Tissue samples were resuspended in 200 μL of 20 mM Tris pH 7.4 containing 2% SDS and were homogenized by sonication in a Branson Digital Sonifier 250 with a 3-mm microtip. Proteins were then extracted and de-crosslinked at 95 °C for 45 min and 65 °C for 4 h. The amount of histones was estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel following protein detection with colloidal Comassie staining (Expedeon) by comparison with known amounts of recombinant histone H3.1 (New England Biolabs). For PAT-H-MS coupled with manual macrodissection or laser microdissction, 10-μm sections were placed on glass slides (Leica Microsystems) and deparaffinized by incubation in xylene (Carlo Erba, Milan, Italy) for 1 min. Tissue sections were subsequently rehydrated in decreasing concentrations of ethanol (100, 95, and 75%) and rinsed in deionized water for 30 s. The slides were stained with hematoxylin for 2 min, washed in deionized water, and dehydrated by incubation in 75% ethanol for 30 s. Each step was performed at room temperature. When using manual macrodissection, the tissue areas corresponding to the tumor xenografts and the normal mouse brain were morphologically evaluated by visualization under a microscope. The two areas, which were clearly identifiable macroscopically, were scraped off the slide with a scalpel into an eppendorf tube, washed once with 1 ml of histolemon to remove any remaining paraffin and rehydrated by a 3 min incubation at room temperature in decreasing concentrations of ethanol (100, 70, 50, 20, and water) followed by a 3 min centrifugation. The tissue was then processed as in the original PAT-H-MS protocol.
PAT-H-MS coupled with LMD
Cancer areas were collected in Eppendorf tubes from tissue sections by laser microdissection, using a Leica LMD 7000 instrument (Leica Microsystems, Wetzlar, Germany) in the “draw and cut” mode with the following laser settings: wavelength 349 nm, pulse energy 2 μJ, numerical aperture 55, speed 15, specimen balance 35, head current 100%, pulse frequency 5000 Hz, and focus offset 65. Tissue pieces were transferred at the bottom of the tubes through a 3-min centrifugation at maximum speed. Tubes were opened carefully to avoid losing tissue pieces and were processed as described above for macrodissected tissue.
A histone-focused super-stable isotope labeling by amino acid in cell culture (SILAC) approach was used as we have recently described . MDA-MB-231, MDA-MB-468, MDA-MB-453, and MDA-MB-361 breast cancer cells lines were grown in SILAC-DMEM (Euroclone) supplemented with 2 mM l-glutamine, 146 mg/l of lysine (Sigma-Aldrich), 84 mg/l l-13C6
15N4-arginine (Arg-10, Sigma-Aldrich), 10% dialyzed serum (Life Technologies), and penicillin/streptomycin for at least eight doublings to obtain complete labeling with heavy-labeled aminoacids. Histones were isolated from the different cell lines through nuclei isolation on a sucrose cushion followed by acidic extraction, as described , mixed in equal amounts, lyophilized, and stored at −80 °C until use.
About 2–5 μg of histones per run per sample were separated on a 17% SDS-PAGE gel and bands corresponding to histone H3 were excised and in-gel digested as previously described . Briefly, gel bands were cut in pieces and de-stained 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 an ultra-nanoflow high-performance liquid chromatography (HPLC) system (EASY-nLC™ 1000, Thermo Fisher Scientic) or an EASY-Spray column (Thermo Fisher Scientic), 50 cm (inner diameter 75 μm, PepMap C18, 2 μm particles), which were connected online to a Q Exactive HF instrument (Thermo Fisher Scientific) through a Nanospray Flex™ or an EASY-Spray™ Ion Sources (Thermo Fisher Scientific), respectively. 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 HF instrument was operated in the data-dependent acquisition (DDA) mode to automatically switch between full-scan MS and tandem mass spectrometry (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 ms for full scans and 50 ms 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.
Acquired RAW data were analyzed by the integrated MaxQuant software v.18.104.22.168, which performed peak list generation and protein identification using the Andromeda search engine . The Uniprot HUMAN_histones 1502 databases was 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. No missed cleavages were allowed, and the minimum peptide length was set to six amino acids. Variable modifications included 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 , the raw data were analyzed through multiple parallel MaxQuant jobs , setting different combinations of variable modifications: (1) D3-acetylation, lysine monomethylation with D3-acetylation, dimethylation, and lysine acetylation, (2) D3-acetylation, lysine monomethylation with D3-acetylation, dimethylation, and trimethylation, and (3) D3-acetylation, lysine monomethylation with D3-acetylation, trimethylation, and lysine acetylation. Peptides with Andromeda scores less than 60 and localization probability scores less than 0.75 were removed. Identifications and retention times were used to guide the manual quantification of each modified peptide using QualBrowser version 2.0.7 (ThermoFisher Scientific). Site assignment was evaluated using QualBrowser and MaxQuant Viewer 22.214.171.124. 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 percent 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 . 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 % RA calculated. L/H ratios of relative abundances were used to compare samples. To better visualize differences among samples, the ratio of one sample relative to the standard was divided by the average ratios across the samples, obtaining “normalized” ratios, which were visualized and clustered using Perseus , with correlation distance and complete linkage as parameters. Differences between luminal A-like and triple negative samples were assessed by t test analysis using GraphPad Prism. For statistical analysis, the ratio for peptides quantitated reliably in the spike-in standard but not in the light channel (encircled grey in Fig. 4a) was considered as 0, while those peptides that could not be quantitated in the heavy channel (grey in Fig. 4a), giving an infinite L/H ratio, were not included in the analysis.