DOI: 10.25345/c56d5pq03
title: MassIVE MSV000097980 - p62 protein pleomorphism confers tissue-specificity
title: Mass spectrometry acquisition for crosslinking peptides Cross-linked peptides were resolved for mass spectrometry analysis using a Dionex Ultimate 3000 RSLCnano liquid chromatography system. Peptides were initially loaded from the autosampler onto an Acclaim PepMap 100 C18 LC Trap Cartridge (0.3 mm inside diameter, 5 mm length) (Thermo Scientific, San Jose, CA) using a loading pump flow rate of 8 ul/minute. Peptides were subsequently resolved for mass spectrometry analysis using an analytical column (75 um inside diameter, 250 mm length) packed in-house with ReproSil-Pur C18-aQ 1.9 um resin (Dr. Masch GmbH, Germany). Chromatography was performed using combinations of buffer A (95% water, 5% acetonitrile, and 0.1% formic acid (v/v/v), pH 2.6), and buffer B (20% water, 80% acetonitrile, and 0.1% formic acid (v/v/v), pH 2.6). The following chromatography steps were performed using a flow rate of 180 nl/minute: (1) 2% B for 25 minutes (column equilibration); (2) a linear gradient from 2% to 10% B over 2 minutes; (3) a linear gradient from 10% to 33% B over 78 minutes; (4) a linear gradient from 33% to 55% B over 12 minutes; (5) a linear gradient from 55% to 95% B over 8 minutes; (6) 95% B for 5 minutes; (7) a linear gradient from 95% B to 2% B over 1 minute; (8) 2% B for 10 minutes. Eluted peptides were analyzed by mass spectrometry using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific, San Jose, CA). An MS3 based method was used for identification of DHSO/DSSO cross-linked peptides as follows: Full MS scans were performed using the Orbitrap mass analyzer (60,000 m/z resolution, 1.6 m/z isolation window, and 375-1500 m/z scan range); The top 3 peptides identified with charge state 4 to 8 were selected for MS2 fragmentation (25% CID energy) and subsequent detection with the Orbitrap mass analyzer (30,000 m/z resolution and a dynamic exclusion time of 40 s); Pairs of MS2 fragments with a mass difference of 31.9720 (10 ppm mass tolerance) were selected for MS3 fragmentation (HCD energy 35%) and detection using the Linear Ion Trap mass analyzer (rapid scan, 3 m/z isolation window, maximum ion injection time 200 ms); Each MS2 scan was followed by a maximum of 4 MS3 scans. In PRM, targeted mass triggers were applied at MS1 using inclusion list for to each FAIMS setting. Data Processing Proteome Discoverer v3.0 (Thermo Fisher Scientific) was used for PTM analysis, with dynamic modifications including phosphorylation on S, T, and Y (+79.996 Da), acetylation on K (+42.011 Da), and M oxidation (+15.995 Da). Crosslinking analysis using DSSO and DHSO was performed with Proteome Discoverer v3.0 integrated with XlinkX v3.0. The detailed searching includes peptide mass (500-10,000 Da); minimum peptide length (5 residues), precursor mass tolerance (10 ppm), product-ion mass tolerance for Orbitrap data (20 ppm), mass tolerance for ion trap data (0.5 Da), modifications (oxidation (M), carbamidomethyl (C)), and 1% FDR control for CSMs. For DSSO cross-links, lysine (K), Serine (S), Threonine (T) and Tyrosine (Y) residues were used for cross-links with a mass shift 158.0038 Da, cleavage alkene fragment mass of 54.0106 Da and thiol fragment mass of 85.9826 Da. For DHSO/DMTMM cross-links, DHSO cross-links were searched with asparanin acid (D) and Glutamine acid (E) for a mass shift 186.0575 Da, cleavage alkene fragment mass of 68.0375 Da and thiol fragment mass of 100.0095 Da. As DMTMM can promote the crosslink reaction between K and D/E, DMTMM non-cleavable cross-links were searched with a mass shift -18.0105 Da. The data were searched against both whole database (NCBI Drosophila Melanogaster reference proteome plus contaminant sequences and p62-sfGFP-Flag sequence) and small database that generated by ID characterization of purified protein samples.