DOI: 10.1002/pmic.200401266. Comparison of protein and peptide prefractionation methods for the shotgun proteomic analysis of <b><i>Synechocystis</i></b> sp. PCC 6803

Comparison of protein and peptide prefractionation methods for the shotgun proteomic analysis of Synechocystis sp. PCC 6803

10.1002/pmic.200401266

Crossref journal-article

Wiley

PROTEOMICS (311)

Abstract

AbstractProteome analysis by gel‐free “shotgun” proteomics relies on the simplification of a peptide mixture before it is analyzed in a mass spectrometer. While separation on a reverse‐phase (RP) liquid chromatographic column is widely employed, a variety of other methods have been used to fractionate both proteins and peptides before this step. We compared six different protein and peptide fractionation workflows, using Synechocystis sp. PCC 6803, a useful model cyanobacterium for potential exploitation to improve its production of hydrogen and other secondary metabolites. Pre‐digestion protein separation was performed by strip‐based isoelectric focusing, one‐dimensional polyacrylamide gel electrophoresis, or weak anion exchange chromatography, while pre‐RP peptide separation was accomplished by isoelectric focusing (IEF) or strong cation exchange chromatography. Peptides were identified using electrospray ionization quadrupole time of flight‐tandem mass spectrometry. Mass spectrometry (MS) and tandem mass spectra were analyzed using ProID software employing both a single organism database and the entire NCBI non‐redundant database, and a total of 776 proteins were identified using a stringent set of selection criteria. Method comparisons were made on the basis of the results obtained (number and types of proteins identified), as well as ease of use and other practical aspects. IEF‐IEF protein and peptide fractionation prior to RP gave the best overall performance.

Bibliography

Gan, C. S., Reardon, K. F., & Wright, P. C. (2005). Comparison of protein and peptide prefractionation methods for the shotgun proteomic analysis of Synechocystis sp.âPCC 6803. PROTEOMICS, 5(9), 2468â2478. Portico.

Authors 3

Chee Sian Gan (first)
Kenneth F. Reardon (additional)
Phillip C. Wright (additional)

References 61 Referenced 77

Dates

Type	When
Created	20 years, 3 months ago (May 9, 2005, 6:47 a.m.)
Deposited	1 year, 11 months ago (Sept. 12, 2023, 9:30 p.m.)
Indexed	1 year, 1 month ago (Aug. 2, 2024, 4:31 a.m.)
Issued	20 years, 3 months ago (June 1, 2005)
Published	20 years, 3 months ago (June 1, 2005)
Published Online	20 years, 3 months ago (June 2, 2005)
Published Print	20 years, 3 months ago (June 1, 2005)

Funders 0

None

BibTeX

@article{Gan_2005, title={Comparison of protein and peptide prefractionation methods for the shotgun proteomic analysis of Synechocystis sp. PCC 6803}, volume={5}, ISSN={1615-9861}, url={http://dx.doi.org/10.1002/pmic.200401266}, DOI={10.1002/pmic.200401266}, number={9}, journal={PROTEOMICS}, publisher={Wiley}, author={Gan, Chee Sian and Reardon, Kenneth F. and Wright, Phillip C.}, year={2005}, month=jun, pages={2468–2478} }

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