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J Am Soc Mass Spectrom. 2017 Aug;28(8):1600-1611. doi: 10.1007/s13361-017-1674-3. Epub 2017 Apr 26.

An EThcD-Based Method for Discrimination of Leucine and Isoleucine Residues in Tryptic Peptides.

Author information

1
Department of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory 1 Bld. 3, 119991, Moscow, Russia.
2
Department of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory 1 Bld. 3, 119991, Moscow, Russia. mocehops@yandex.ru.

Abstract

An EThcD-based approach for the reliable discrimination of isomeric leucine and isoleucine residues in peptide de novo sequencing procedure has been proposed. A multistage fragmentation of peptide ions was performed with Orbitrap Elite mass spectrometer in electrospray ionization mode. At the first stage, z-ions were produced by ETD or ETcaD fragmentation of doubly or triply charged peptide precursor ions. These primary ions were further fragmented by HCD with broad-band ion isolation, and the resulting w-ions showed different mass for leucine and isoleucine residues. The procedure did not require manual isolation of specific z-ions prior to HCD stage. Forty-three tryptic peptides (3 to 27 residues) obtained by trypsinolysis of human serum albumin (HSA) and gp188 protein were analyzed. To demonstrate a proper solution for radical site migration problem, three non-tryptic peptides were also analyzed. A total of 93 leucine and isoleucine residues were considered and 83 of them were correctly identified. The developed approach can be a reasonable substitution for additional Edman degradation procedure, which is still used in peptide sequencing for leucine and isoleucine discrimination. Graphical Abstract ᅟ.

KEYWORDS:

EThcD; Leucine/isoleucine differentiation; Orbitrap; Peptide sequencing; Tryptic peptides

PMID:
28447219
DOI:
10.1007/s13361-017-1674-3
[Indexed for MEDLINE]

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