Identification of 2D-gel proteins: A comparison of MALDI/TOF peptide mass mapping to μ LC-ESI tandem mass spectrometry

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and μLC-MS/MS is presented. 100% of the ge...

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Veröffentlicht in:	Journal of the American Society for Mass Spectrometry 2003-09, Vol.14 (9), p.957-970
Hauptverfasser:	Lim, Hanjo, Eng, Jimmy, Yates, John R, Tollaksen, Sandra L, Giometti, Carol S, Holden, James F, Adams, Michael W.W, Reich, Claudia I, Olsen, Gary J, Hays, Lara G
Format:	Artikel
Sprache:	eng
Schlagworte:	Amino Acid Sequence Analytical, structural and metabolic biochemistry Biological and medical sciences Chromatography, Liquid Databases, Protein Electrophoresis, Gel, Two-Dimensional Fundamental and applied biological sciences. Psychology General aspects, investigation methods Methanococcus - chemistry Molecular Sequence Data Molecular Weight Peptide Mapping - methods Proteins Proteins - analysis Proteins - chemistry Pyrococcus furiosus - chemistry Sensitivity and Specificity Software Spectrometry, Mass, Electrospray Ionization - methods Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods Trypsin
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Zusammenfassung:	A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and μLC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by μLC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip C18 and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (
ISSN:	1044-0305 1879-1123
DOI:	10.1016/S1044-0305(03)00144-2