Breast cancer sample collection
In our study, 52 human breast cancer tissue specimens and 3 para-cancer tissue samples were collected from breast cancer patients who underwent surgical resections in China Medical University. (Shenyang, China). Para-cancer tissues are non-cancerous that are obtained from > 2 cm from the tumor margin. All tissue specimens were retained for pathologic examination with HE staining to determine the pathologic type and other auxiliary tests, such as immunohistochemical and FISH testing. The remaining tissue specimens were washed with cold phosphate buffered saline (GE Healthcare, Beijing, China) to remove the residual blood after surgical resection, then immediately frozen in liquid nitrogen and stored at -80℃ for further analysis. None of the 52 breast cancer patients received pre-operative radiation, chemotherapy, or other treatments. This study was conducted in accordance with the principles expressed in the Helsinki declaration and approved by the Research Ethics Committee of China Medical University. (IRB approval number:AF-SOP-07-1.1-01). Informed consent was obtained from all patients. A summary of clinical and pathologic data for the breast cancer patients is shown in Supplementary Table 1 (red data represent 3 TNBC samples and green data represent 3 luminal A breast cancer samples).
Experimental procedures and strategies
Fifty-five samples were established by Data Dependent Acquisition(DDA) mass spectrometry. Then, based on the constructed database, DIA relative quantitative proteomics analysis was performed on 3 TNBC samples and 3 luminal A breast cancer samples to compare the differences between the two groups and screen out differentially expressed proteins. After that, we continued to perform IPA-based bioinformatics analysis of differentially expressed proteins.
Sample preparation
The tissue was scraped and added with an appropriate amount of SDT lysate, then transferred to a Lysing Matrix A tube. The homogenizer was used for homogenization crushing (24×2, 6.0 m/S, 60s, twice). After ultrasound, the homogenized tissue was placed in a boiling water bath for 10 min, then centrifuged at 14000g for 15 min. The supernatant was filtered through a 0.22-m centrifuge tube and the filtrate was collected. Protein was quantified using the BCA method. The samples were packed and stored at -80℃.
SDS-PAGE electrophoresis
Protein (20 µg) was taken from each sample and added to a 6X sample loading buffer, followed by a boiling water bath for 5 min, separation on 12% SDS-PAGE (constant pressure 250V for 40 min), and staining with Coomassie brilliant blue.
FASP enzyme [6]
A 200-ug protein solution was taken from each sample. DTT was added to a final concentration of 100mM and the solution was placed in a boiling water bath for 5 min, then the samples were cooled to room temperature. Two hundred micron of UA buffer was added to the protein solution and mixed well, then transferred to a 30kD ultrafiltration centrifuge tube and centrifuged at 12,500g for 25 min. The filtrate was discarded (this step was repeated twice). One hundred microliters of IAA buffer in UA was added, oscillated at 600 rpm for 1 min, reacted at room temperature against light for 30 min, and centrifuged at 12,500g for 25 min. One hundred microliters of UA buffer was added and centrifuged at 12,500g for 15 min (this step was repeated twice). One hundred microliters of 0.1M TEAB solution was added and centrifuged at 12,500g for 15 min (this step was repeated twice). Forty microliters of trypsin buffer (4μL g of trypsin in 40 mu L of 0.1M TEAB solution) was oscillated at 600 rpm for 1 min, and placed at 37℃ for 16-18 h. The collection tube was replaced, centrifuged at 12,500g for 15 min, 20μL of 0.1M TEAB solution was added, centrifuged at 12500g for 15 min, and the filtrate was collected. The peptide fragment was desalted with a C 18 cartridge, freeze-dried, and re-dissolved with 40μL of 0.1% formic acid solution. The peptide fragment was quantified.
High PH RP classification
All the peptide compounds were classified using an Agilent 1260 Infinity II HPLC system. Buffer solution A was 10mM HCOONH4 and 5% can (pH 10.0) and buffer solution B was 10mM HCOONH4 and 85% can (pH 10.0).The column was balanced with liquid A and the samples were separated from the column (Waters, XBridge Peptide BEH C18 Column, 130A, 5 m, 4.6 mm X 100 mm) with a flow rate of 1 mL/min. The liquid phase gradient was as follows: using a linear gradient, the column temperature was maintained at 30℃ within 40 min from 5% B to 45% B. Thirty-six components were collected and dried in a vacuum concentrator. After freeze-drying, the samples were re-dissolved with 0.1% formic acid solution in 12 fractions.
DDA mass spectrometry library
Six microliters were taken from each fraction and added to 1 ul of 10× iRT peptide fragment. After mixing, 6 ul of sample was injected, separated by nano-lc, and analyzed by on-line electrospray tandem mass spectrometry. The whole liquid-mass series system was as follows: 1) liquid phase system, Easy nLC system (Thermo Fisher Scientific); 2) mass spectrometry system, q-exactive hf-x (Thermo Fisher Scientific). Buffer solution A was 0.1% formic acid solution and solution B was 0.1% acetonitrile solution (acetonitrile was 80%). The samples were separated by a gradient of non-linear growth in an analytical column (Thermo Fisher Scientific, Acclaim PepMap RSLC 50 um X 15 cm, nano viper, P/N164943) at a flow rate of 300 nL/min for 0-5 min, 1% B for 5-95 min, 1% B to 28% B for 95-110 min, 28% B to 38% B for 110-115 min, 38% B to 100% B for 115-120 min, and 100% B. The electrospray voltage was 2.0kV.
The mass spectrum parameters were set as follows: (1) MS, scan range (m/z)=350–1500; resolution=60,000; AGC target=3e6; maximum injection time=30 ms; included charge states=2-7; filter dynamic exclusion: exclusion duration=30s; and (2) dd-MS2, isolation window=1.6 m/z; resolution=15,000; AGC target=1e5; maximum injection time=45 ms; and NCE=28%.
The Spectronaut Pulsar X (version 12, Biognosys AG) was used to consolidate and analyze the original mass spectrometry data and establish a spectrograph database. The database was Swissprot_human_isoform_201806 (42356 entries), as below: 2018-06, download link: http://www.uniprot.org. Trypsin enzymolysis was set to allow two missing cutting sites. Carbamidomethyl (C), variable modification: Oxidation (M), and acetyl (Protein n-term) n-terminal acetylation. The standard for database construction was 1% precursor false discovery rate( FDR), 1% protein FDR, and 1% peptide FDR.
DIA mass spectrometry
Six samples (TNBC and luminal A breast cancer [3 cases each]) were removed and 6 ul was added to 1 ul of 10× iRT peptide, mixed, separated with nano-lc, and analyzed by on-line electrospray tandem mass spectrometry. The whole liquid-mass series system was as follows: 1) liquid phase system, Easy nLC system (Thermo Fisher Scientific); and 2) mass spectrometry system, q-exactive hf-x (Thermo Fisher Scientific). Buffer solution A was 0.1% formic acid solution and solution B was 0.1% acetonitrile solution (acetonitrile was 80%). The samples were separated by a gradient of non-linear growth in the analytical column (Thermo Fisher Scientific, Acclaim PepMap RSLC 50um X 15cm,nano viper, P/N164943) at a flow rate of 300 nL/min for 0-5 min, 1% B for 5-95 min, 1% B to 28% B for 95-110 min, 28% B to 38% B for 110-115 min, 38% B to 100% B for 115-120 min, and 100% B. The electrospray voltage was 2.0kV.
The mass spectrum parameters were set as follows: (1) MS, scan range (m/z)=350–1500; resolution=60,000; AGC target=3e6; maximum injection time=50 ms; and (2) DIA, resolution=15,000; AGC target=2e5; maximum injection time=45ms; and NCE=28%.
Bioinformatics Analyses
Differential proteins performed IPA-based classical pathway analysis, upstream regulation analysis, disease and function analysis, regulation effect analysis, and interaction network analysis. A detailed description of these analyses is provided in the supplementary information.