A simplified method for peptide de novo sequencing using O-18
labeling

J 2014

A simplified method for peptide de novo sequencing using O-18 labeling

VORÁČ, Aleš, Ondrej ŠEDO, Jan HAVLIŠ and Zbyněk ZDRÁHAL

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

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10406 Analytical chemistry

Country of publisher

United Kingdom of Great Britain and Northern Ireland

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

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

Abstract

V originále

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 project

Name: CEITEC - central european institute of technology

GBP206/12/G151, research and development project

Name: Centrum nových přístupů k bioanalýze a molekulární diagnostice

Citovat

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, 2014, vol. 20, No 3, p. 255-260. ISSN 1469-0667. Available from: https://dx.doi.org/10.1255/ejms.1277.

@article{1194851,
   author = {Voráč, Aleš and Šedo, Ondrej and Havliš, Jan and Zdráhal, Zbyněk},
   article_location = {CHICHESTER (ENGLAND)},
   article_number = {3},
   doi = {http://dx.doi.org/10.1255/ejms.1277},
   keywords = {peptide de novo sequencing; mass spectrometry; isotopic labeling; O-18 incorporation},
   language = {eng},
   issn = {1469-0667},
   journal = {European Journal of Mass Spectrometry},
   title = {A simplified method for peptide de novo sequencing using O-18 labeling},
   url = {https://www.impublications.com/content/abstract?code=E20_0255},
   volume = {20},
   year = {2014}
}

TY  - JOUR
ID  - 1194851
AU  - Voráč, Aleš - Šedo, Ondrej - Havliš, Jan - Zdráhal, Zbyněk
PY  - 2014
TI  - A simplified method for peptide de novo sequencing using O-18 labeling
JF  - European Journal of Mass Spectrometry
VL  - 20
IS  - 3
SP  - 255-260
EP  - 255-260
PB  - IM PUBLICATIONS
SN  - 14690667
KW  - peptide de novo sequencing
KW  - mass spectrometry
KW  - isotopic labeling
KW  - O-18 incorporation
UR  - https://www.impublications.com/content/abstract?code=E20_0255
L2  - https://www.impublications.com/content/abstract?code=E20_0255
N2  - 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.
ER  -

VORÁČ, Aleš, Ondrej ŠEDO, Jan HAVLIŠ and Zbyněk ZDRÁHAL. A simplified method for peptide de novo sequencing using O-18 labeling. \textit{European Journal of Mass Spectrometry}. CHICHESTER (ENGLAND): IM PUBLICATIONS, 2014, vol.~20, No~3, p.~255-260. ISSN~1469-0667. Available from: https://dx.doi.org/10.1255/ejms.1277.

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