| Metadata |
| datasetIdentifier | PASS00857 |
| datasetType | Other |
| submitter | Olga Schubert <olga.schubert@gmail.com> |
| submitter_organization | ETHZ |
| lab_head_full_name | Pavel Bouchal |
| lab_head_email | bouchal@chemi.muni.cz |
| lab_head_organization | Masaryk University |
| lab_head_country | Czech Republic |
| datasetTag | BreastCancerLibrary |
| datasetTitle | SWATH library for breast cancer studies |
| publicReleaseDate | 2019-07-23 00:00:00 |
| finalizedDate | 2019-07-24 21:40:09 |
| summary | Accurate classification of breast tumors is vital for patient management decisions and enables more precise cancer treatment. Here, we present a quantitative proteotyping approach based on sequential windowed acquisition of all theoretical fragment ion spectra (SWATH) mass spectrometry and establish key proteins for breast tumor classification. The study is based on 96 tissue samples representing five conventional breast cancer subtypes. SWATH proteotype patterns largely recapitulate these subtypes; however, they also reveal varying heterogeneity within the conventional subtypes, with triple negative tumors being the most heterogeneous. Proteins that contribute most strongly to the proteotypebased classification include INPP4B, CDK1, and ERBB2 and are associated with estrogen receptor (ER) status, tumor grade status, and HER2 status. Although these three key proteins exhibit high levels of correlation with transcript levels (R > 0.67), general correlation did not exceed R = 0.29, indicating the value of protein-level measurements of disease regulated genes. Overall, this study highlights how cancer tissue proteotyping can lead to more accurate patient stratification. |
| contributors | Pavel Bouchal, Olga T. Schubert, Jakub Faktor, Lenka Capkova, Hana Imrichova, Karolina Zoufalova, Vendula Paralova, Roman Hrstka, Yansheng Liu, H. Alexander Ebhardt, Eva Budinska, Rudolf Nenutil and Ruedi Aebersold |
| publication | Bouchal, P. et al. (2019). Breast Cancer Classification Based on Proteotypes Obtained by SWATH Mass Spectrometry. Cell Reports 28, 832–843.e837. PubMed ID: 31315058 |
| growth | Informed patient consent forms along with tissue procurement procedures were approved by the ethics committee of the Masaryk Memorial Cancer Institute (MMCI). Breast cancer tissues were frozen in liquid nitrogen within 20 minutes after surgical removal and stored at -180°C in the tissue bank at MMCI. A set of 96 preoperatively untreated breast carcinomas of 11-20 mm maximum diameter (pT1c) was selected for the study. It consisted of 48 estrogen receptor (ER) positive, progesterone receptor (PR) positive, HER2 negative, grade 1 (luminal A-like (LA)) tumors; 16 ER positive, PR positive or negative, HER2 negative, grade 3 (luminal B-like (LB) tumors); 8 ER positive, PR positive or negative, HER2 positive, grade 3 (luminal B-like HER2 positive (LBH) tumors); and 16 ER negative, PR negative, HER2 negative, grade 3 (triple negative-like (TN) tumors). Half of the tumors in each group were lymph node positive and half were lymph node negative at the time of diagnosis. |
| treatment | na |
| extraction | Frozen breast cancer tissue (approx. 20 mm3) was homogenized in 150 μl lysis buffer (6 M guanidine hydrochloride; 0.1 M Na-phosphate buffer, pH 6.6; 1% Triton X-100) in MM301 mechanic homogenizer (Retsch, Germany) using a metal ball for 2 × 2 min at 20 s-1, needle-sonicated (Bandelin 2200 Ultrasonic homogenizer, Bandelin, Germany; 30 × 0.1 s pulses at 50 W) and kept on ice for 1 h. After centrifugation, protein concentration was measured in the supernatant using RC-DC assay (Bio-Rad, USA). |
| separation | As an input for the SWATH-MS assay library, the following peptide samples were prepared: (i) 10 pooled samples (each pooled from 4-8 patients) of 5 the breast cancer immunophenotypes mentioned above, each immunophenotype involved a pair of lymph node positive and lymph node negative cases; (ii) pool of aliquots of all samples in the sample set (400 µg in total) fractionated using HILIC chromatography as follows: HILIC Kinetex column (Phenomenex, USA, 2.6 µm, 150 x 2.1 mm, 100 A) was accommodated in Agilent Infinity 1260 LC system (Agilent, USA), mobile phase (A) was composed of 100% acetonitrile (Merck, Germany), mobile phase (B) of water (MilliQ, Millipore) and mobile phase (C) of 50 mM ammonium formate (pH 3.2). 20 µL mobile phase (B) were added to the sample which was then sonicated on ultrasonic bath for 2 min. Then, 20 µL mobile phase (A) and 5 µL mobile phase (C) were added. After a further 2 min sonication, the sample was centrifuged at 16,000 x g at 20 °C for 20 min. The sample injection volume was 40 µL and the separation method was set as follows: 5 min isocratic 0% B, 7 min gradient to 20% B, 23 min gradient to 34% B, 5 min gradient to 50% B, 5 min isocratic 50% B, 0.5 min gradient to 0% B and for 4.5 min isocratic 0% B; mobile phase C was kept at 10% all the time. The flow rate was 0.2 mL/min, column temperature was set to 30 °C and the signal was monitored at 280 nm. Fractions were collected every 1 min, some neighboring fractions with lower signal intensity were pooled together to obtain final 20 fractions with similar peptide content. These were vacuum-dried and stored at -80°C. |
| digestion | The lysate (volume corresponding to 60 μg of protein) was digested using a filter aided sample preparation protocol. Briefly, aliquots of the lysate were mixed with 200 μl 8 M urea in 0.5 M triethylammonium bicarbonate (TEAB) pH 8.5 on Vivacon 500 filter device, cut-off 10K (Sartorius Stedim Biotech GmbH, Germany). The device was centrifuged at 14 000 × g at 20°C for 20 min (all of the following centrifugation steps were performed applying the same conditions). Subsequently, 100 μl 5 mM tris(2-carboxyethyl)phosphine in 8 M urea, 0.5 M TEAB, pH 8.5 was added to the filter, proteins were reduced at 37°C for 60 min at 600 rpm and centrifuged. Next, 100 μl 10 mM S-methyl methanethiosulfonate in 8 M urea and 0.5 M TEAB, pH 8.5 were added to the filter, cysteine groups of peptides were alkylated at 20°C for 10 min and centrifuged. The resulting concentrate was diluted with 100 μl 8 M urea in 0.5 M TEAB, pH 8.5 and concentrated again. This step was repeated twice. The concentrate was subjected to proteolytic digestion by adding 100 μl 0.5 M TEAB, pH 8.5 containing trypsin (TPCK treated, AB Sciex, USA) reconstituted in water (trypsin to protein weight ratio 1:30) and by incubating at 37°C for 16 h. The digests were collected by centrifugation into clean tubes, dried in a vacuum concentrator and C18 desalted using 0.1% trifluoracetic acid as an ion pairing reagent. Eleven retention time anchor peptides (iRT peptides, Biognosys AG, Zurich, Switzerland) were added into each sample at a ratio of 1:40 v/v. |
| acquisition | MS/MS datasets for spectral library generation were acquired on TripleTOF 5600+ mass spectrometer (AB SCIEX, Canada) interfaced to an Eksigent Ekspert nanoLC 400 system (AB SCIEX, Canada). Prior to separation, the peptides were concentrated on a C18 PepMap100 pre-column (Thermo Scientific, USA; particle size 5 µm, 100 Å, 300 µm x 5 mm). After 10 min washing using 2% acetonitrile containing 0.05% (v/v) trifluoroacetic acid, the peptides were eluted on a capillary column (75 μm × 250 mm, X-Bridge BEH C18 130 Å, particle size 2.5 μm, Waters, USA) using 2% mobile phase B for 10 min (mobile phase A was composed of 0.1% (v/v) formic acid in water, mobile phase B of 0.1% (v/v) formic acid in acetonitrile) followed by gradient elution from 2% to 40% mobile phase B in the next 120 minutes at a flow rate of 300 nl/min. Output of the separation column was directly coupled to nano-electrospray source. MS1 spectra were collected in the range of 400-1250 m/z for 250 ms. The 20 most intense precursors with charge states of 2 to 5 that exceeded 50 counts per second were selected for fragmentation, rolling collision energy was used for fragmentation and MS2 spectra were collected in the range of 200–1600 m/z for 100 ms. The precursor ions were dynamically excluded from reselection for 12 s. |
| informatics | Raw data files (wiff) were centroided and converted into mzML format using the AB Sciex converter (beta release 111102) and subsequently converted into mzXML using openMS (version 1.9.0, Feb 10 2012, Revision 9534). The converted data files were searched using the search engines X!Tandem (k-score, version 2011.12.01.1) and Comet (version 2013.02, revision 2) against all human proteins annotated in UniProt/SwissProt (2014_04) and the sequences of 11 iRT peptides (iRT-kit, Biognosys), as well as for every of these target proteins a decoy protein based on reversed protein sequences. Only fully tryptic peptides with up to two missed cleavages were allowed for the database search. The tolerated mass errors were 15 ppm on MS1 level and 0.1 Da on MS2 level. Methylthiolation of cysteines was defined as a fixed modification and methionine oxidation as a variable modification. The search results were processed with PeptideProphet and iProphet as part of the TPP 4.6.0. The SWATH-MS assay library was constructed from the iProphet results with an iProphet cut-off of 0.8360 which corresponds to 1% FDR on peptide level. The raw and consensus spectral libraries were built with SpectraST (version 4.0) using the -cICID_QTOF option for high resolution and high mass accuracy. Retention times were normalized and converted to iRT space using spectrast2spectrast_iRT.py (imsproteomicstools R356). The 6 most intense y and b fragment ions of charge state 1, 2 and 3 were extracted from the consensus spectral library using spectrast2tsv.py (imsbproteomicstools). Neutral losses -17 (NH3), -18 (H2O) and -64 (CH4SO, typical for oxidized methionine) were also included if they were among the 6 most intense fragment ions. Fragment ions falling into the SWATH window of the precursor were excluded as the resulting signals are often highly interfered. The library was converted into TraML format using the OpenMS tool ConvertTSVToTraML (version 1.10.0). Decoy transition groups were generated based on shuffled sequences (decoys similar to targets were excluded) by the OpenMS tool OpenSwathDecoyGenerator (version 1.10.0) and appended to the final SWATH library in TraML format. |
| instruments | TripleTOF 5600+ |
| species | Human |
| massModifications | static: C+45.987721 |
