Ltq orbitrap elite hybrid mass spectrometer

The peptides were analyzed by an online LC-MS system. They were separated by reversed-phase liquid chromatography (LC), and the LC is coupled to an LTQ Orbitrap Elite Hybrid Mass Spectrometer (Thermo Scientific). MS/MS analysis was performed with a data-dependent Top20 method. The detailed information is in Supporting Information.
Raw MS files were analyzed by MaxQuant (version1.6.2.3).^{28 (link)} MS spectra were searched against the human proteome database downloaded from UniProt using the integrated Andromeda search engine.²⁹ All default parameters were left unchanged, except adding variable modification for glycosylation site identification (+2.9883 Da) and 3 maximum missed cleavages. Label-free quantification was also enabled with the LFQ min ratio count of 1, the match-between-runs option was enabled, and the iBAQ option was enabled. The false discovery rates (FDRs) were kept at 0.01 at the peptide spectrum match, protein, and site decoy fraction levels.

Eluted peptides were purified, and dried peptides were subsequently dissolved in a solution with 5% ACN and 4% formic acid (FA) prior to LC-MS/MS analysis. Peptides were separated by a Dionex UltiMate 3000 UHPLC system (Thermo Scientific), which is coupled to an LTQ Orbitrap Elite Hybrid Mass Spectrometer (Thermo Scientific) with Xcalibur software (version 3.0.63). A total of ~1 µg of peptides was loaded onto a C18-packed microcapillary column (Magic C18AQ, 3 μm, 200 Å, 100 μm × 16 cm, Michrom Bioresources) by a Dionex WPS-3000TPL RS autosampler (Thermostatted Pulled Loop Rapid Separation Nano/Capillary Autosampler). Peptides were separated by reversed-phase chromatography using an UltiMate 3000 binary pump with a 90-minute gradient of 4–30% ACN containing 0.125% FA. MS/MS analysis was performed with a data-dependent Top20 method. For each cycle, a full MS scan (resolution: 60,000) in the Orbitrap with 1 million automatic gain control (AGC) target was followed by up to 20 MS/MS in the LTQ for the most intense ions.^{55 (link), 56 (link)} Selected ions were excluded from further sequencing for 90 seconds. Ions with single or unassigned charge were not sequenced. Maximum ion accumulation times were 1,000 ms for each full MS scan and 50 ms for MS/MS scans.