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

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• 作者：Elena L. Rudashevskaya ; Florian P. Breitwieser ; Marie L. Huber ; Jacques Colinge ; Andr茅 C. M眉ller ; Keiryn L. Bennett
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：February 5, 2013
• 年：2013
• 卷：85
• 期：3
• 页码：1454-1461
• 全文大小：388K
• 年卷期：v.85,no.3(February 5, 2013)
• ISSN：1520-6882

文摘

The identification and validation of cross-linked peptides by mass spectrometry remains a daunting challenge for protein鈥損rotein 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 (BSp>3p>). On the basis of these results, MSDAM was created and assessed on the BSp>3p>-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.