Open AccessIdentification of 2D-gel proteins: A comparison of MALDI/TOF peptide mass mapping to μ LC-ESI tandem mass spectrometry
Author(s) -
H. Lim,
Jimmy K. Eng,
John R. Yates,
Sandra L. Tollaksen,
Carol S. Giometti,
James F. Holden,
Michael W. W. Adams,
Claudia I. Reich,
Gary J. Olsen,
Lara G. Hays
Publication year - 2003
Publication title -
journal of the american society for mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.961
H-Index - 127
eISSN - 1879-1123
pISSN - 1044-0305
DOI - 10.1016/s1044-0305(03)00144-2
Subject(s) - chemistry , mass spectrometry , chromatography , peptide mass fingerprinting , tandem mass spectrometry , bottom up proteomics , peptide , molecular mass , peptide sequence , pyrococcus furiosus , spots , protein mass spectrometry , matrix assisted laser desorption/ionization , proteomics , biochemistry , archaea , enzyme , desorption , organic chemistry , adsorption , gene
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 mu LC-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 mu LC-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 (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.