Mass Spectrometric Analysis

Mass spectrometric analysis of the Glu-C derived peptides was performed by LC-MS-MS on a linear ion trap-orbitrap hybrid mass spectrometer (Orbitrap-Classic, Thermo) equipped with a nanoelectrospray ion source (Thermo) and coupled to a Proxeon EASY-nLC system. Peptides were injected onto a Thermo (Part No. 160321) Acclaim PepMap100 reverse phase C₁₈ 3μm column, 75μm × 15 cm, with a flow of 300 nl/min and eluted with a 45 min linear gradient of 95% solvent A (2% Acetonitrile, 0.1% formic acid in H₂O) to 35% solvent B (90% acetonitrile, 0.08% formic acid in H₂O) at 20 min followed by a rise to 80%B at 23 min, maintained at 80% B for 5 min, followed by re-equilibration. The instrument was operated with the “lock mass” option to improve the mass accuracy of precursor ions and data were acquired in the data-dependent mode, automatically switching between MS and MS-MS acquisition. Full scan spectra (m/z 340–1800) were acquired in the orbitrap with resolution R = 60,000 at m/z 400 (after accumulation to an FTMS Full AGC Target; 1,000,000; MSn AGC Target; 100,000). The 5 most intense ions, above a specified minimum signal threshold (5,000), based upon a low resolution (R = 15,000) preview of the survey scan, were fragmented by collision induced dissociation and recorded in the linear ion trap, (Full AGC Target; 30,000. MSn AGC Target; 5,000). Multi-Stage-Activation was used to provide a pseudo MS3 scan of any parent ions showing a neutral loss of 48.9885, 32.6570, 24.4942, allowing for 2+, 3+ and 4+ ions respectively. The resulting pseudo MS3 scan was automatically combined with the relevant MS2 scan prior to data analysis. RAW files containing data from the Orbitrap-Classic were analysed directly by using Proteome Discoverer 1.4 and phosphoRS 3.1 (Thermo), utilising Mascot (www.matrixscience.com), and searching against an in house database containing the relevant sequences.