GoKawiilCollection of tissue samples
The autopsy samples involved in this study were collected from the body donation centre of Dalian Medical University and Shanghai Jiao Tong University. The post-mortem interval was within 10 h. The project was approved by the Ethics Committee of Dalian Medical University (institutional review board (IRB): Dalian Medical 2019-05) and Shanghai Jiao Tong University (IRB: 2022-A-01, 2021-03). Written informed consent was obtained from the legally authorized representatives for all donors. To ensure anatomical accuracy, we used (1) macrodissection by two experienced anatomists (included in the author list) following a standardized protocol; and (2) histopathological validation through H&E-stained formalin-fixed paraffin-embedded (FFPE) sections reviewed independently by two pathologists (included in the author list) for tissue types that might not be clearly partitioned at the anatomical level.
Paired tumours and non-tumour samples from surgery were collected from Harbin Medical University Cancer Hospital (from December 2019 to December 2022) and Huashan Hospital (from January 2018 to December 2018). The project was approved by the Ethics Committee of Harbin Medical University Cancer Hospital (IRB: KY2019-08, KY2023-03 and KY2019-16) and Huashan Hospital (KY2021-064). Written informed consent for each patient was collected. The samples mentioned above were stored in FFPE format. We systematically compared the cancer types, number and types of samples of our pan-cancer cohort with those in TCGA15 and CPTAC49. According to the NCI Genomic Data Commons50, our study includes two rare malignancies not covered by either TCGA or CPTAC: GIST and fallopian tube carcinoma. Conversely, TCGA and CPTAC include several cancer types absent in our current cohort, including adrenal cortical carcinoma, bladder cancer, leukaemia, mesothelioma, skin cancers and melanoma. As regards sample size, our study profiles approximately 40 patients per cancer type (Supplementary Table 1), whereas TCGA typically includes around 200 cases per type and CPTAC includes about 160. With respect to normal control tissues, TCGA mainly uses blood samples for germline genomic analysis, whereas CPTAC often includes matched adjacent non-tumour tissues (either fully or partially paired). In our pan-cancer cohort, we incorporated matched adjacent non-tumour tissues for most patients, allowing tumour–non-tumour comparisons at the proteome level.
Autopsy specimens of fetuses, from abortions, were collected from Shenzhen Baoan District Maternal and Child Health Hospital (during 16 June 2020 to 16 September 2020). The post-mortem interval was within 10 h. The project was approved by the Ethics Committee of Shenzhen Baoan District Maternal and Child Health Hospital (IRB: LLSCHY 2019-10-36). Written informed consent was obtained from all donors of tissues. The fetal samples were stored in formalin at 4 °C before use.
Sample preparation for LC–MS
The sample preparation process was similar to that described previously51,52,53. In brief, we punched each FFPE sample and weighed about 1.0 mg. Heptane was used for dewaxing, gradient (100%, 90%, 75%) ethanol for hydration and 0.1% formic acid (FA) for acid hydrolysis. The obtained product was then placed in pressure cycling technology (PCT) microtubes, and Tris-HCl (pH 10, freshly prepared) was added at 95 °C for 30 min for alkaline hydrolysis. After rapid cooling, lysis buffer (6 M urea, 2 M thiourea), Tris(2-carboxyethyl)phosphine (TCEP) and iodoacetamide (IAA) were added for reductive alkylation. PCT-assisted lysis was performed in the Barocycler NEP2320-Enhanced (Pressure BioSciences) for 90 cycles at 30 °C, with each cycle consisting of 30 s at high pressure (45,000 psi/~310 MPa) followed by 10 s at ambient pressure (AP). LysC (enzymatic substrate concentration ratio 1:80, Hualishi Tech) and trypsin (enzymatic substrate concentration ratio 1:20, sequencing grade, Hualishi Tech) were then added for digestion. The process was conducted with PCT for 120 cycles at 30 °C, with each cycle consisting of 50 s at high pressure (20,000 psi/~138 MPa) followed by 10 s at AP. After the enzymatic hydrolysis process was terminated by 10% trifluoroacetic acid (TFA), we used C18 spin columns (The Nest Group) for desalination.
The dried samples were resuspended with buffer A (high-performance liquid chromatography (HPLC)-grade water, pH 10). The fractionation was performed using an UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) with an HPLC C18 column (diameter 4.6 mm, length 25 cm, particle size 300 Å). The flow rate was 0.5 μl per min. The gradient was 5% to 95% buffer B (98% acetonitrile (ACN), pH 10) in 60 min. A total of 60 fractions were collected. Then, 60 fractionations were combined into 10. The combined samples were evaporated using vacuum centrifugation (CentriVap, Labconco) at 45 °C.
For bone tissues, we cut a piece approximately one-fifth of the volume of a 2 ml centrifuge tube. The teeth were shattered with a clean hammer and placed into the 2 ml tube. After the dewaxing and alkaline hydrolysis described above, we added lysis buffer and grinding beads (about one-third of the volume of the 2 ml tube). The sample was frozen in liquid nitrogen, ground in a tissue grinder for 1 min and refrozen in liquid nitrogen. This procedure was repeated three to five times until the sample turned into a bone slurry (except for the teeth). We then added 1 ml of 10% FA solution and incubated the sample at 4 °C overnight for digestion. Afterwards, approximately 4 mg of the insoluble wet weight was transferred to a PCT tube, and 150 µl Tris-HCl (pH 10) was added to neutralize any excess acid, followed by removal of the supernatant. Then, 20 µl Tris-HCl (pH 10) and 30 µl 6 M urea–2 M thiourea lysis buffer were added, and subsequent PCT-assisted lysis and enzymatic digestion were performed as described above.
Hair samples (4 cm) or nail samples (1 mg) were immersed in 50% methanol or ethanol, chopped, and vortexed, then transferred to a PCT tube. To the tube, 30 µl lysis buffer (30% trifluoroethanol, Tris-HCl buffer, pH 8) and 2.5 µl of 200 mM TCEP solution were added, and the tube was sealed with a PCT pestle and placed in a PCT device. The samples underwent 180 cycles in the Barocycler (50 s at 45,000 psi (~310 MPa), 10 s at atmospheric pressure, 70 °C). After cooling to room temperature, 2.5 µl of 800 mM IAA solution was added, and the samples were incubated in the dark at room temperature for 30 min with gentle mixing (800 rpm). After dilution with 150 µl Tris-HCl buffer (pH 8), 1.25 µg of LysC and 5 µg of trypsin were added, and the tube was sealed and subjected to 120 cycles in the PCT (20,000 psi (~138 MPa) for 50 s, atmospheric pressure for 10 s, 30 °C). After the cycles, 15 µl of 10% TFA solution was added to stop the digestion, and the samples were desalted using C18 tips.
Whole blood was collected by venipuncture into commercial EDTA-containing sampling containers. It was separated into PRP1, erythrocyte, platelet-rich plasma (PRP), platelet-free plasma (PPP) and the pure platelet fraction, according to a previously published article54. PRP1 was the supernatant in the first centrifuge at 200g for 10 min. PRP was the supernatant in the centrifuge of PRP1 at 200g for 10 min. Then, 1 ml whole blood was transferred from the anticoagulation tube into a 50 ml centrifuge tube, 10 ml 1× red blood cell lysate (Invitrogen, 00-4333) was added for 10 min at room temperature (strictly controlled time) and 25 ml phosphate-buffered saline (PBS; total volume 36 ml) was added to stop the reaction. It was then divided into 15 ml centrifuge tubes (9 ml in each tube) and centrifuged at 500g for 15 min at 4 °C, followed by aspirating and discarding the supernatant. At this time, 200 μl was allowed to remain in each 15 ml centrifuge tube. The remaining liquid and cell pellet from each tube were pipetted into a 1.5 ml centrifuge tube, which was centrifuged at 500g for 5 min at 4 °C, followed by aspirating the supernatant as much as possible, to leave the cell pellet. The cell pellet was washed with 1× PBS three times and centrifuged (500g for 5 min, 4 °C) to get a clean white blood cell pellet.
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