Reversed phase c18 spe columns

Approximately 100 mg of each of the cryopulverized breast cancer xenograft tissues^{43 (link)} were sonicated in 1.5 mL of lysis buffer (8 M urea, 0.8 M NH₄HCO₃, pH 8.0). Protein concentrations of the tissue lysates were determined by a BCA assay (Pierce, Rockford, IL). Proteins were reduced with 10 mM TCEP for 1 h at 37 °C and subsequently alkylated with 12 mM iodoacetamide for 1 h at room temperature in dark. Samples were then diluted 1:4 with deionized water and digested with sequencing-grade modified trypsin (Promega, Madison, WI) at a 1:50 enzyme-to-substrate ratio. After 12 h of digestion at 37 °C, another aliquot of the same amount of trypsin was added to the samples and the samples were further incubated at 37 °C overnight. The digested samples were then acidified with 10% trifluoroacetic acid to pH <3. Tryptic peptides were desalted on reversed-phase C18 SPE columns (Waters, Milford, MA) and dried using a Speed-Vac.

The experimental design is shown in Figure 1A. Approximately 30–200 mg of each of the sectioned ovarian tumor tissues or non-tumor tissues were homogenized separately in lysis buffer (8 M urea, 1.0 M NH₄HCO₃, pH 8.0) by sonication (Branson Sonifier 250, 15 s cycles with 1 min cool down, 4 times, 20% output). Lysates were precleared by centrifugation at 16,500 g for 15 min at 4°C and protein concentrations were determined by BCA assay (Pierce). Proteins (2mg/mL) were reduced with 10 mM tris (2-carboxyethyl) phosphine (TCEP) for 1 h at 37°C, and subsequently alkylated with 15mM iodoacetamide for 1 h at room temperature (RT) in the dark. Samples were diluted 1:5 with deionized water and digested with sequencing grade modified trypsin (Promega) at a 1:50 enzyme-to-substrate ratio. After overnight digestion at 37°C, another aliquot of the same amount of trypsin was added to the samples and further incubated at 37°C overnight. The digested samples were then acidified with 50% trifluoroacetic acid (TFA, Sigma) to ~pH 2. Tryptic peptides were desalted on reversed phase C18 SPE columns (Waters) and dried using a Speed-Vac (Thermo Scientific).

Dried peptides from each sample were labeled with 11-plex TMT (Tandem Mass Tag) reagents (Thermo Fisher Scientific). Peptides (300 μg) from each of the HNSCC and NAT samples were dissolved in 60 μL of 50 mM HEPES, pH 8.5 solution. An internal quality control (QC) sample, NCI-7 Cell Line (Clark et al., 2018 (link)), was interspersed among all TMT 11-plex sets. HNSCC and NAT samples with NCI-7 QC aliquots were co-randomized to 19 TMT sets. A reference sample was created by pooling an aliquot from 87 HNSCC tissues and 50 NAT tissues (representing ~80% of the sample cohort), and included in all TMT 11-plex sets as a pooled reference channel. Five mg of TMT reagent was dissolved in 250 μL of anhydrous acetonitrile, and then 20 μL of each TMT reagent was added to the corresponding aliquot of peptides. After 1h incubation at RT, the reaction was quenched by incubation with 5% NH2OH for 15 min at RT. Following labeling, peptides were desalted on reversed phase C18 SPE columns (Waters) and dried using Speed-Vac (Thermo Scientific).