Unblocking the Sink: Improved CID-Based Analysis of Phosphorylated Peptides by Enzymatic Removal of the Basic <i>C</i>-Terminal Residue

Lanucara, Francesco, Lee, Dave Chi Hoo and Eyers, Claire E ORCID: 0000-0002-3223-5926 (2014) Unblocking the Sink: Improved CID-Based Analysis of Phosphorylated Peptides by Enzymatic Removal of the Basic <i>C</i>-Terminal Residue. JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY, 25 (2). pp. 214-225.

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Abstract

A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H3PO4), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H3PO4 elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H3PO4 upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available.

Item Type:	Article
Uncontrolled Keywords:	Collision-induced dissociation, Phosphorylation, Mobile proton, Dissociation mechanisms, Tandem mass spectrometry, Ion trap mass spectrometry, Carboxypeptidase-B
Depositing User:	Symplectic Admin
Date Deposited:	18 Jun 2015 16:30
Last Modified:	17 Oct 2023 01:00
DOI:	10.1007/s13361-013-0770-2
Publisher's Statement :	© The Author(s), 2013. This article is published with open access at Springerlink.com. This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/2014061

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