VORÁČ, Aleš, Ondrej ŠEDO, Jan HAVLIŠ and Zbyněk ZDRÁHAL. A simplified method for peptide de novo sequencing using O-18 labeling. European Journal of Mass Spectrometry. CHICHESTER (ENGLAND): IM PUBLICATIONS, vol. 20, No 3, p. 255-260. ISSN 1469-0667. doi:10.1255/ejms.1277. 2014.
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Basic information
Original name A simplified method for peptide de novo sequencing using O-18 labeling
Authors VORÁČ, Aleš (203 Czech Republic, belonging to the institution), Ondrej ŠEDO (203 Czech Republic, belonging to the institution), Jan HAVLIŠ (203 Czech Republic, belonging to the institution) and Zbyněk ZDRÁHAL (203 Czech Republic, guarantor, belonging to the institution).
Edition European Journal of Mass Spectrometry, CHICHESTER (ENGLAND), IM PUBLICATIONS, 2014, 1469-0667.
Other information
Original language English
Type of outcome Article in a journal
Field of Study 10406 Analytical chemistry
Country of publisher United Kingdom of Great Britain and Northern Ireland
Confidentiality degree is not subject to a state or trade secret
WWW URL
Impact factor Impact factor: 1.000
RIV identification code RIV/00216224:14740/14:00073785
Organization unit Central European Institute of Technology
Doi http://dx.doi.org/10.1255/ejms.1277
UT WoS 000337268400006
Keywords in English peptide de novo sequencing; mass spectrometry; isotopic labeling; O-18 incorporation
Tags kontrola MP, rivok
Tags International impact, Reviewed
Changed by Changed by: Martina Prášilová, učo 342282. Changed: 11/3/2015 11:30.
Abstract
Incorporation of an O-18 atom into a peptide C-terminus by proteolytic cleavage in the presence of (H2O)-O-18 is one of the most effective ways of enhancing tandem mass spectrometry (MS/MS)-based de novo sequencing. Incorporation is usually accomplished by procedures including vacuum-assisted drying of tryptic peptides extracted from gels, their subsequent reconstitution in a (H2O)-O-16/(H2O)-O-18 mixture and re-treatment with trypsin. In the present work, we propose a simplified procedure for O-18 incorporation into tryptic peptides by adding (H2O)-O-18 and trypsin to the original digest solution. In comparison to published methods, the proposed protocol for peptide de novo sequencing brings significant advantages in analysis and workflow with no deterioration in method performance. We show that labeling by this simplified method leads to a highlighting of the y-ion fragment series in the peptide matrix-assisted laser desorption/ionization (MALDI)-MS/MS data, which facilitates MS/MS data interpretation. We also prove that eliminating acid extraction of peptides from gels does not result in a decrease in sequence coverage or a qualitative loss of particular peptides detectable by MALDI-MS. The method was examined by MALDI-MS/MS on bovine serum albumin and recombinant histidine kinase CKI1 from Arabidopsis thaliana, and was verified by de novo sequencing of tryptic peptides originating from Apodemus sylvaticus salivary proteins.
Links
ED1.1.00/02.0068, research and development projectName: CEITEC - central european institute of technology
GBP206/12/G151, research and development projectName: Centrum nových přístupů k bioanalýze a molekulární diagnostice
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