title: Improved peptide identification by targeted fragmentation using CID, HCD and ETD on an LTQ-Orbitrap Velos.
abstract: Over the past decade peptide sequencing by collision induced dissociation (CID) has become the method of choice in mass spectrometry-based proteomics. The development of alternative fragmentation techniques such as electron transfer dissociation (ETD) has extended the possibilities within tandem mass spectrometry. Recent advances in instrumentation allow peptide fragment ions to be detected with high speed and sensitivity (e.g., in a 2D or 3D ion trap) or at high resolution and high mass accuracy (e.g., an Orbitrap or a ToF). Here, we describe a comprehensive experimental comparison of using ETD, ion-trap CID, and beam type CID (HCD) in combination with either linear ion trap or Orbitrap readout for the large-scale analysis of tryptic peptides. We investigate which combination of fragmentation technique and mass analyzer provides the best performance for the analysis of distinct peptide populations such as N-acetylated, phosphorylated, and tryptic peptides with up to two missed cleavages. We found that HCD provides more peptide identifications than CID and ETD for doubly charged peptides. In terms of Mascot score, ETD FT outperforms the other techniques for peptides with charge states higher than 2. Our data shows that there is a trade-off between spectral quality and speed when using the Orbitrap for fragment ion detection. We conclude that a decision-tree regulated combination of higher-energy collisional dissociation (HCD) and ETD can improve the average Mascot score.
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Christian K
authors.initials: CK
authors.lastname: Frese
authors.forename: A F Maarten
authors.initials: AF
authors.lastname: Altelaar
authors.forename: Marco L
authors.initials: ML
authors.lastname: Hennrich
authors.forename: Dirk
authors.initials: D
authors.lastname: Nolting
authors.forename: Martin
authors.initials: M
authors.lastname: Zeller
authors.forename: Jens
authors.initials: J
authors.lastname: Griep-Raming
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
authors.forename: Shabaz
authors.initials: S
authors.lastname: Mohammed
mesh_terms: D002460:Cell Line; D006801:Humans; D010455:Peptides / Q000235:genetics*; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010455:Peptides