Pierce c18 spin tips

Approximately 5 mg of WMP was suspended in 100 μl trifluoroacetic acid (TFA) and incubated at room temperature for 10 min (30 (link)). Samples were further irradiated for 10 s at 800 W using a microwave oven. Samples were neutralized with 2M TrisBase using 10× volume relative to TFA before adding DTT to 10 mM and reducing for 30 min at 37 °C. Iodoacetamide was added to a 2.5× molar excess over DTT, and samples were incubated in the dark for 15 min. Digestion was carried out for 20 h at 37 °C using trypsin at an enzyme:protein ratio of 1:50. Aliquots of digested samples containing 10 μg of protein were desalted using Pierce C18 Spin Tips (Thermo Scientific #84850) according to the manufacturer's protocol.

The proteins on the silicone slices were reduced by adding 200 µL of 10 mM dithiothreitol in ABC buffer (100 mM of ammonium-bicarbonate, pH 8.0), followed by incubation at room temperature for 1 h. For protein digestion, 2 µg of trypsin was added, and the reaction was agitated overnight at 37 °C. The alkylation of free cysteines was performed by adding 20 µL of 550 mM iodoacetamide in ABC buffer for 20 min in the dark. The supernatants were transferred to clean low-binding tubes, followed by desalting with C18 Tips (Pierce C18 Spin Tips, P/N: 87784, Thermo). The desalted peptides were dried and stored at −20 °C.

A2780, A2780-cis, IGROV-1, and IGROV-1-cis cells with 80% confluence were harvested into cold 1 × PBS by a scraper and washed three times by 1 × PBS. The protein samples were prepared according to our previous study (9 (link)). A total of 100 μg protein was precipitated in 100% ice-cold acetone at 1:4 (v/v), incubated with trypsin at an enzyme/substrate ratio of 1:50 (w/w) at 37°C overnight, and then eluted using Pierce™ C18 Spin Tips (ThermoFisher Scientific, VIC, Australia).

To identify a protein composition in E. coli lysates, proteins from 5 μl of clarified lysates were precipitated with acetone. Four volumes of acetone chilled to −20 °C were added to clarified lysates followed by 1 h of incubation at −20 °C. Precipitates were collected by 10 min of centrifugation at 20,000g at 4 °C. Pellets were rinsed with 80% acetone and let dry in air for 15 min. Dried pellets were dissolved in 5 μl of 50 mM NH₄HCO₃, 8 M urea, then mixed with 50 μl of 50 mM NH₄HCO₃ containing 20 ng μl⁻¹ SOLu trypsin (Sigma) and incubated overnight at 25 °C.
After the overnight incubation, digestion reactions were mixed with an equal volume 2% HFBA, incubated at room temperature for 5 min and clarified by 5 min of centrifugation at 16,000g. Peptides from supernatants were then desalted using Pierce C18 spin tips (Thermo) according to the manufacturer’s instructions and dried under a vacuum. Dried peptides were reconstituted in 0.1% formic acid and concentration was measured at 205 nm on a Nanodrop One (Thermo).

PNGase F digestion was performed on a 10 kDa cutoff filter (Sartorius #VN01H02) prior to FASP digestion. Filters were first equilibrated with successive washes of 0.1% FA followed by 8M urea, 100 mM ABC pH 8.0. All washes were spun through at 14,000 × g for 15 min. Samples were then loaded onto filters and spun at 14,000 × g for 20 min before washing with 200 μl of 8M urea, 100 mM ABC. Filters were then washed with three aliquots of 100 μl 50 mM ABC pH 8.0 before adding 1000U PNGase F (NEB #P0704) in 2M urea, 100 mM ABC pH 8.0 directly to the top of the filter membrane. PNGase F digestion was allowed to proceed for 2 h at 37 °C before spinning at 14,000 × g for 15 min to remove digest volume, retaining deglycosylated protein on the membrane. Filters were then washed with 100 μl 50 mM ABC pH 8.0 to remove residual buffer. Trypsin digest was performed in 20 mM ABC pH 8.0 with 0.02% ProteaseMax (Promega #V207 A) for 16 h using a 1:100 enzyme:protein ratio. Samples were eluted and acidified in 150 μl 0.2% FA. Aliquots of digested samples containing 10 μg of protein were desalted using Pierce C18 Spin Tips (Thermo Scientific #84850) according to the manufacturer's protocol.

Peptides were desalted using C-18 stage tips according to Rappsilber et al., 2007 (link). C-18 material (three C-18 plugs were pasted in a 200 μl pipette tip, Pierce C18 Spin Tips, 84,850 Thermo Scientific, Bishop’s Stortford, United Kingdom) was equilibrated with methanol/ 0.1% FA, 70% ACN/ 0.1% FA and with 0.1% FA. Peptides were loaded on C-18 material, washed with 0.1% FA and eluted with 70% ACN/0.1% FA. Samples were dried and finally, peptides were resuspended in 20 μl 0.1% FA. For glycopeptide enrichment, peptides were first desalted using Poros oligo r3 resin (1-339-09, Thermo Scientific, Bishop’s Stortford, United Kingdom) as described (Gobom et al., 1999 (link); Queiroz et al., 2019 (link)). Pierce centrifuge columns (SH253723, Thermo Scientific, Bishop’s Stortford, United Kingdom) were filed with 250 μl of Poros oligo r3 resin. Columns were washed three times with 0.1% TFA. Peptides were loaded onto the columns and washed three times with 0.1% TFA and subsequently eluted with 70% ACN.