An EThcD-Based Method for Discrimination of Leucine and Isoleucine Residues in Tryptic Peptides
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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.
KeywordsLeucine/isoleucine differentiation Orbitrap EThcD Tryptic peptides Peptide sequencing
The authors are thankful to Thermo Fisher Scientific Inc., Textronica AG group (Moscow, Russia), and personally to Professor Alexander Makarov for providing Orbitrap Elite mass spectrometer for this work. They also express their gratitude to Professor Valery Shevchenko (Cancer Research Center, Moscow), Dr. Irina Tarasova (Insitute for Energy Problems of Chemical Physics, Russian Academy of Sciences), Dr. Ludmila Alekseeva and Dr. Lidia Kurochkina (Institute of Bioorganic Chemistry, Russ. Acad. Sci.) for proteins and synthetic peptides used in this studies.
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