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    2020-08-18

    r> METHOD DETAILS
    Tissue quality control via RNA integrity measurement
    After homogenization in a MM301 mechanical homogenizer (Retsch, Germany) using a metal ball for 2 3 2 min at 25 s-1 in 600 mL of RLT buffer (QIAGEN, Germany) with 1% b-mercaptoethanol, total RNA was isolated using RNeasy Mini Kit (QIAGEN, Germany) following the manufacturer’s protocol. RNA was eluted with 30 mL of RNase-free water, quantified at 260 nm using NanoDrop ND-1000 (Thermo Fisher Scientific, USA) and quality checked by measurement of RNA integrity number (RIN) on Agilent 2100 Bioanalyzer using RNA 6000 Nano Kit (both Agilent Technologies, USA). Samples which did not pass the criterion of RNA quality
    (RIN > 7) were excluded and replaced by other tissues with the same clinicopathological characteristics for the SWATH-MS experiments.
    Proteomics sample preparation
    Frozen breast cancer tissue (approx. 20 mm3) was homogenized in 150 mL lysis buffer (6 M guanidine hydrochloride; 0.1 M Na-phosphate buffer, pH 6.6; 1% Triton X-100) in a MM301 mechanic homogenizer (Retsch, Germany) using a metal ball for 2 3 2 min at 20 s-1, needle-sonicated (Bandelin 2200 Ultrasonic homogenizer, Bandelin, Germany; 30 3 0.1 s pulses at 50 W) and kept on ice for 1 h. After 14,000 x g centrifugation at 4 C for 20 min, protein concentration was measured in the supernatant using RC-DC assay (Bio-Rad, USA). An aliquot of the lysate containing 60 mg total protein mass was digested using a filter aided sample preparation protocol (Wisniewski et al., 2011) with modifications. Briefly, aliquots of the lysate were mixed with 200 mL 8 M urea in 0.5 M triethylammonium bicarbonate (TEAB) pH 8.5 on Vivacon 500 filter device, cut-off 10K (Sartorius Stedim Biotech, Germany). The device was centrifuged at 14,000 3 g at 20 C for 20 min (all of the following centrifugation steps were performed applying the same conditions). Subsequently, 100 mL 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 mL 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 mL 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 FUB-NPB-22 by adding 100 mL 0.5 M TEAB, pH 8.5 containing trypsin (TPCK treated, 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 as previously described (Bouchal et al., 2009) using 0.1% trifluoracetic acid as an ion pairing reagent. Eleven retention time anchor peptides (commercial iRT peptide solution, Biognosys, Switzerland) (Escher et al., 2012) were added into each sample at a ratio of 1:40 v/v. For SWATH-MS analysis, equal amounts of samples (estimated to be 1.33 mg protein) were injected in single technical replicates.
    LC-MS analyses for spectral library generation
    As an input for generating the SWATH-MS assay library, the following samples were prepared: (i) 10 pooled samples (each pooled from 4-8 patients) of 5 the breast cancer subtypes mentioned above. Each subtype group involved two pools of tumors (lymph node positive and lymph node negative cases separately); (ii) pool of aliquots of all samples in the sample set (400 mg in total) fractionated using HILIC chromatography as follows: HILIC Kinetex column (Phenomenex, USA, 2.6 mm, 150 3 2.1 mm, 100 A) was run in an Agilent Infinity 1260 LC system (Agilent, USA). Mobile phase (A) was composed of 100% acetonitrile (Merck, Germany), mobile phase
    10% all the time. The flow rate was 0.2 mL/min, column temperature was set to 30 C and the UV signal was monitored at 280 nm. Fractions were collected every 1 min, some neighboring fractions with lower signal intensity were subsequently pooled
    to generate a final set of 20 fractions with similar peptide content. These were vacuum-dried and stored at 80 C.
    MS/MS datasets for spectral library generation were acquired on a TripleTOF 5600+ mass spectrometer (SCIEX, Canada)
    interfaced to an Eksigent Ekspert nanoLC 400 system (SCIEX, Canada). Prior to separation, the peptides were concentrated on a C18 PepMap100 pre-column (Thermo Fisher Scientific, USA; particle size 5 mm, 100 A˚, 300 mm x 5 mm). After 10 min washing with a solvent consisting of 2% acetonitrile and 0.05% (v/v) trifluoroacetic acid, the peptides were eluted from a capillary column (75 mm 3 250 mm, X-Bridge BEH C18 130 A˚, particle size 2.5 mm, Waters, USA, prepared as described in (Planeta et al., 2003)) 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 min 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.