title: Trimethylation enhancement using diazomethane (TrEnDi): rapid on-column quaternization of peptide amino groups via reaction with diazomethane significantly enhances sensitivity in mass spectrometry analyses via a fixed, permanent positive charge.
abstract: Defining cellular processes relies heavily on elucidating the temporal dynamics of proteins. To this end, mass spectrometry (MS) is an extremely valuable tool; different MS-based quantitative proteomics strategies have emerged to map protein dynamics over the course of stimuli. Herein, we disclose our novel MS-based quantitative proteomics strategy with unique analytical characteristics. By passing ethereal diazomethane over peptides on strong cation exchange resin within a microfluidic device, peptides react to contain fixed, permanent positive charges. Modified peptides display improved ionization characteristics and dissociate via tandem mass spectrometry (MS(2)) to form strong a2 fragment ion peaks. Process optimization and determination of reactive functional groups enabled a priori prediction of MS(2) fragmentation patterns for modified peptides. The strategy was tested on digested bovine serum albumin (BSA) and successfully quantified a peptide that was not observable prior to modification. Our method ionizes peptides regardless of proton affinity, thus decreasing ion suppression and permitting predictable multiple reaction monitoring (MRM)-based quantitation with improved sensitivity.
authors.affiliation: Department of Chemistry, Carleton University , Ottawa, Ontario K1S 5B6, Canada.
authors.forename: Karl V
authors.initials: KV
authors.lastname: Wasslen
authors.forename: Le Hoa
authors.initials: le H
authors.lastname: Tan
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Manthorpe
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Smith
mesh_terms: D000588:Amines / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D003978:Diazomethane / Q000737:chemistry*; D057230:Limit of Detection; D008745:Methylation; D010455:Peptides / Q000737:chemistry*; D040901:Proteomics; D012710:Serum Albumin, Bovine / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000588:Amines; D010455:Peptides; D012710:Serum Albumin, Bovine; D003978:Diazomethane