Rhode Island Hospital COBRE Proteomics Core facility performed all mass spectrometry analysis by nano-LC-ESI-MS/MS using an Ultimate3000 nano-LC system (Dionex) controlled with Chromeleon software coupled to a QSTAR XL (Applied Biosystems, Concord, Ontario, CA) mass spectrometer. Tryptic digests were fractionated by reverse-phase chromatography using a C-18 PepMap 100 column (75 um id x 15 mm, 3um particle size, LC Packings/Dionex, Sunnyvale, CA) operating at a flow of 300 nL/min. Over a 40 min time period, a linear separation gradient was applied starting at 5 % (v/v) CAN in 0.1 % (v/v) formic acid (Buffer A) to 95 % (v/v) ACN in 0.1 % (v/v) formic acid (Buffer B). ESI was used to introduce the column elutate directly into the mass spectrometer.
Candidate ions were selected and fragmented using a standard information dependent acquisition (IDA) method. During MS/MS scans, one-second MS scans (range between 350 and 1800 Thompson, Thompson (Th) = Da/z) were used to identify candidates for fragmentation. MS/MS scans (2 s; range between 150 and 1800 Th) were collected up to three times after each survey scan. Candidates considered for fragmentation required an assigned charge in the range of +2 or +4.
Candidate ions were selected and fragmented using a standard information dependent acquisition (IDA) method. During MS/MS scans, one-second MS scans (range between 350 and 1800 Thompson, Thompson (Th) = Da/z) were used to identify candidates for fragmentation. MS/MS scans (2 s; range between 150 and 1800 Th) were collected up to three times after each survey scan. Candidates considered for fragmentation required an assigned charge in the range of +2 or +4.