title: Multiple and sequential data acquisition method: an improved method for fragmentation and detection of cross-linked peptides on a hybrid linear trap quadrupole Orbitrap Velos mass spectrometer.
abstract: The identification and validation of cross-linked peptides by mass spectrometry remains a daunting challenge for protein-protein cross-linking approaches when investigating protein interactions. This includes the fragmentation of cross-linked peptides in the mass spectrometer per se and following database searching, the matching of the molecular masses of the fragment ions to the correct cross-linked peptides. The hybrid linear trap quadrupole (LTQ) Orbitrap Velos combines the speed of the tandem mass spectrometry (MS/MS) duty circle with high mass accuracy, and these features were utilized in the current study to substantially improve the confidence in the identification of cross-linked peptides. An MS/MS method termed multiple and sequential data acquisition method (MSDAM) was developed. Preliminary optimization of the MS/MS settings was performed with a synthetic peptide (TP1) cross-linked with bis[sulfosuccinimidyl] suberate (BS(3)). On the basis of these results, MSDAM was created and assessed on the BS(3)-cross-linked bovine serum albumin (BSA) homodimer. MSDAM applies a series of multiple sequential fragmentation events with a range of different normalized collision energies (NCE) to the same precursor ion. The combination of a series of NCE enabled a considerable improvement in the quality of the fragmentation spectra for cross-linked peptides, and ultimately aided in the identification of the sequences of the cross-linked peptides. Concurrently, MSDAM provides confirmatory evidence from the formation of reporter ions fragments, which reduces the false positive rate of incorrectly assigned cross-linked peptides.
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: Elena L
authors.initials: EL
authors.lastname: Rudashevskaya
authors.forename: Florian P
authors.initials: FP
authors.lastname: Breitwieser
authors.forename: Marie L
authors.initials: ML
authors.lastname: Huber
authors.forename: Jacques
authors.initials: J
authors.lastname: Colinge
authors.forename: André C
authors.initials: AC
authors.lastname: Müller
authors.forename: Keiryn L
authors.initials: KL
authors.lastname: Bennett
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000235:genetics; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000235:genetics; D013223:Statistics as Topic / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010446:Peptide Fragments; D012710:Serum Albumin, Bovine