The mass spectrometry proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE [87 (link)] partner repository with the dataset identifier PXD034057.
From each sample, total proteins were proteolyzed with LysC (Wako Chemicals, Neuss, Germany) and trypsin (Promega) using a suspension trapping protocol (S-Trap, Protifi) to remove SDS according to the manufacturer’s instructions. Briefly, samples were reduced and carbamidomethylated, followed by acidification with phosphoric acid and addition of methanol to a final concentration of >70% before loading to the trap columns. Proteins were washed while trapped on column, then digested on column with LysC (2 hours at RT) followed by trypsin (overnight, at 37 C). Peptides were collected by centrifugation and acidified. Eluted peptides were analyzed on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) in the data dependent mode. Approximately 0.5 g peptides per sample were automatically loaded to the online coupled ultra-high-performance liquid chromatography (UHPLC) system (Ultimate 3000, Thermo Fisher Scientific). A nano trap column was used (300- m ID X 5mm, packed with Acclaim PepMap100 C18, 5 m, 100 Å; LC Packings) before separation by reversed phase chromatography (Acquity UHPLC M-Class HSS T3 Column 75 m ID X 250 mm, 1.8 m; Waters) at 40 C. Peptides were eluted from the column at 250 nL/min using increasing ACN concentrations (in 0.1% formic acid) from 3% to 41% over a linear 95-min gradient. MS spectra were recorded at a resolution of 60 000 with an AGC target of 3 106 and a maximum injection time of 50 ms from 300 to 1’500 m/z. From the MS scan, the 10 most abundant peptide ions were selected for fragmentation via HCD with a normalized collision energy of 28, an isolation window of 1.6 m/z, and a dynamic exclusion of 30 s. MS/MS spectra were recorded at a resolution of 15’000 with an AGC target of 105 and a maximum injection time of 50 ms. Unassigned charges and charges of +1 and above +8 were excluded from precursor selection.
From each sample, total proteins were proteolyzed with LysC (Wako Chemicals, Neuss, Germany) and trypsin (Promega) using a suspension trapping protocol (S-Trap, Protifi) to remove SDS according to the manufacturer’s instructions. Briefly, samples were reduced and carbamidomethylated, followed by acidification with phosphoric acid and addition of methanol to a final concentration of >70% before loading to the trap columns. Proteins were washed while trapped on column, then digested on column with LysC (2 hours at RT) followed by trypsin (overnight, at 37 C). Peptides were collected by centrifugation and acidified. Eluted peptides were analyzed on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) in the data dependent mode. Approximately 0.5 g peptides per sample were automatically loaded to the online coupled ultra-high-performance liquid chromatography (UHPLC) system (Ultimate 3000, Thermo Fisher Scientific). A nano trap column was used (300- m ID X 5mm, packed with Acclaim PepMap100 C18, 5 m, 100 Å; LC Packings) before separation by reversed phase chromatography (Acquity UHPLC M-Class HSS T3 Column 75 m ID X 250 mm, 1.8 m; Waters) at 40 C. Peptides were eluted from the column at 250 nL/min using increasing ACN concentrations (in 0.1% formic acid) from 3% to 41% over a linear 95-min gradient. MS spectra were recorded at a resolution of 60 000 with an AGC target of 3 106 and a maximum injection time of 50 ms from 300 to 1’500 m/z. From the MS scan, the 10 most abundant peptide ions were selected for fragmentation via HCD with a normalized collision energy of 28, an isolation window of 1.6 m/z, and a dynamic exclusion of 30 s. MS/MS spectra were recorded at a resolution of 15’000 with an AGC target of 105 and a maximum injection time of 50 ms. Unassigned charges and charges of +1 and above +8 were excluded from precursor selection.
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