Development of an LC-MALDI method for the analysis of protein complexes.
In this study, a two-dimensional LC-MALDI-TOF/TOF method has been developed for analyzing protein complexes. In our hands, the method has proven to be an excellent strategy for the analysis of protein complexes isolated in pull-down experiments. This is in part because the preservation of the chromatographic separation on a MALDI target yields an "unlimited" amount of time to obtain MS/MS spectra, making it possible to probe more deeply into complex samples. A brief statistical analysis was performed on the data obtained from the LC-MALDI-TOF/TOF system in order to better understand peptide fragmentation patterns under high-energy collision conditions. These statistical analyses provided some insight into how to evaluate the quality and accuracy of the database search results derived from the TOF/TOF-based analysis. The potential of the method was demonstrated by the successful identification of all the known penicillin-binding proteins in E. coli isolated using a drug-based pull-down with ampicillin as the bait. The performance of the LC-MALDI-TOF/TOF system was compared with that of an equivalent 2D LC-ESI-MS/MS approach, in the analysis of a protein bait-based pull-down. Regardless of the number of peptides identified in the ESI versus MALDI approach, the two approaches were found to be complementary. When the data is merged at the peptide level, the combined result gives higher Mascot scores and an overall higher confidence in protein identification than with either approach alone.
Zhen
Yuejun
Y
Prolexys Pharmaceuticals, Inc., Salt Lake City, Utah 84116, USA.
Xu
Nafei
N
Richardson
Bonnie
B
Becklin
Robert
R
Savage
Justin R
JR
Blake
Kelly
K
Peltier
John M
JM
D000667:Ampicillin / Q000378:metabolism; D002412:Cations / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D002412:Cations; D046911:Macromolecular Substances; D011506:Proteins; D000667:Ampicillin