title: Opposite Electron-Transfer Dissociation and Higher-Energy Collisional Dissociation Fragmentation Characteristics of Proteolytic K/R(X)nand (X)nK/R Peptides Provide Benefits for Peptide Sequencing in Proteomics and Phosphoproteomics.
abstract: A key step in shotgun proteomics is the digestion of proteins into peptides amenable for mass spectrometry. Tryptic peptides can be readily sequenced and identified by collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD) because the fragmentation rules are well-understood. Here, we investigate LysargiNase, a perfect trypsin mirror protease, because it cleaves equally specific at arginine and lysine residues, albeit at the N-terminal end. LysargiNase peptides are therefore practically tryptic-like in length and sequence except that following ESI, the two protons are now both positioned at the N-terminus. Here, we compare side-by-side the chromatographic separation properties, gas-phase fragmentation characteristics, and (phospho)proteome sequence coverage of tryptic (i.e., (X)nK/R) and LysargiNase (i.e., K/R(X)n) peptides using primarily electron-transfer dissociation (ETD) and, for comparison, HCD. We find that tryptic and LysargiNase peptides fragment nearly as mirror images. For LysargiNase predominantly N-terminal peptide ions (c-ions (ETD) and b-ions (HCD)) are formed, whereas for trypsin, C-terminal fragment ions dominate (z-ions (ETD) and y-ions (HCD)) in a homologous mixture of complementary ions. Especially during ETD, LysargiNase peptides fragment into low-complexity but information-rich sequence ladders. Trypsin and LysargiNase chart distinct parts of the proteome, and therefore, the combined use of these enzymes will benefit a more in-depth and reliable analysis of (phospho)proteomes.
authors.forename: Liana
authors.initials: L
authors.lastname: Tsiatsiani
authors.forename: Piero
authors.initials: P
authors.lastname: Giansanti
authors.forename: Richard A
authors.initials: RA
authors.lastname: Scheltema
authors.forename: Henk
authors.initials: H
authors.lastname: van den Toorn
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, and Department of Biochemistry and Molecular Biology, University of British Columbia , Vancouver V6T 1Z3, BC, Canada.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Overall
authors.forename: A F Maarten
authors.identifier: 0000-0001-5093-5945
authors.initials: AF
authors.lastname: Altelaar
authors.forename: Albert J R
authors.identifier: 0000-0002-2405-4404
authors.initials: AJ
authors.lastname: Heck
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004583:Electrons; D007700:Kinetics; D045726:Metalloproteases / Q000737:chemistry*; D010446:Peptide Fragments / Q000032:analysis*; D010750:Phosphoproteins / Q000737:chemistry*; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D000072417:Protein Domains; D059748:Proteolysis; D040901:Proteomics / Q000379:methods; D011522:Protons; D020539:Sequence Analysis, Protein; D013816:Thermodynamics; D014357:Trypsin / Q000737:chemistry*
chemical_list: D010446:Peptide Fragments; D010750:Phosphoproteins; D011522:Protons; D045726:Metalloproteases; C512490:ulilysin, Methanosarcina acetivorans; D014357:Trypsin
keywords: LysargiNase; electron-transfer dissociation; higher-energy collisional dissociation; peptide fragmentation; phosphoproteomics; proteomics; trypsin