title: Mass spectral enhanced detection of Ubls using SWATH acquisition: MEDUSA--simultaneous quantification of SUMO and ubiquitin-derived isopeptides.
abstract: Protein modification by ubiquitination and SUMOylation occur throughout the cell and are responsible for numerous cellular functions such as apoptosis, DNA replication and repair, and gene transcription. Current methods for the identification of such modifications using mass spectrometry predominantly rely upon tryptic isopeptide tag generation followed by database searching with in vitro genetic mutation of SUMO routinely required. We have recently described a novel approach to ubiquitin and SUMO modification detection based upon the diagnostic a' and b' ions released from the isopeptide tags upon collision-induced dissociation of reductively methylated Ubl isopeptides (RUbI) using formaldehyde. Here, we significantly extend those studies by combining data-independent acquisition (DIA) with alternative labeling reagents to improve diagnostic ion coverage and enable relative quantification of modified peptides from both MS and MS/MS signals. Model synthetic ubiquitin and SUMO-derived isopeptides were labeled with mTRAQ reagents (Δ0, Δ4, and Δ8) and subjected to LC-MS/MS with SWATH acquisition. Novel diagnostic ions were generated upon CID, which facilitated the selective detection of these modified peptides. Simultaneous MS-based and MS/MS-based relative quantification was demonstrated for both Ub and SUMO-derived isopeptides across three channels in a background of mTRAQ-labeled Escherichia coli digest.
authors.affiliation: CRUK Manchester Institute, University of Manchester, Manchester, M20 4BX, UK, john.griffiths@cruk.manchester.ac.uk.
authors.forename: John R
authors.initials: JR
authors.lastname: Griffiths
authors.forename: Navin
authors.initials: N
authors.lastname: Chicooree
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Connolly
authors.forename: Milla
authors.initials: M
authors.lastname: Neffling
authors.forename: Catherine S
authors.initials: CS
authors.lastname: Lane
authors.forename: Thomas
authors.initials: T
authors.lastname: Knapman
authors.forename: Duncan L
authors.initials: DL
authors.lastname: Smith
mesh_terms: D053000:Analytic Sample Preparation Methods; D002851:Chromatography, High Pressure Liquid; D055664:Electrochemical Techniques; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011492:Protein Hydrolysates / Q000737:chemistry / Q000378:metabolism; D012815:Signal Processing, Computer-Assisted; D058207:Sumoylation; D053719:Tandem Mass Spectrometry; D057149:Ubiquitinated Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D009842:Oligopeptides; D010455:Peptides; D011492:Protein Hydrolysates; D057149:Ubiquitinated Proteins