Comparative identification of prostanoid inducible proteins by LC-ESI-MS/MS and MALDI-TOF mass spectrometry.
Protein identification by MS is well-established. Mixtures of proteins from cell extracts are separated by either one- or two-dimensional gel electrophoresis, and specific bands or spots are subjected to in-gel digestion and subsequent analysis by MS. The two most common types of ionization used in MS are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). When ESI is used, the sample is typically analyzed by inline HPLC-ESI-MS/MS with fragmentation of individual digest peptides, followed by database comparison between theoretical and experimental fragmentation patterns. MALDI-MS analysis is based on peptide mass mapping, with mass measurements of the digest peptides searched against a database of theoretical digests. We give here the results of a comparison between ESI-ion trap and MALDI-TOF (time-of-flight) analysis of 11-deoxy,16,16-dimethyl prostaglandin E(2) (DDM-PGE(2)) inducible proteins. Individual peptides identified by the two techniques differed, in general, but the resulting protein identification was the same. Slightly higher coverage of each protein was obtained by MALDI-TOF, but the MS/MS data were more definitive by requiring fewer peptides to assign a positive identification. Both methods effectively identified two proteins in the same gel band. The samples here are derived from a renal epithelial cell line (LLC-PK(1)) established from the New Hampshire minipig, a species poorly represented in the current database, and strategies and limitations for analyzing such species are discussed.
Person
Maria D
MD
Center for Molecular and Cellular Toxicology, Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712, USA.
Lo
Herng-Hsiang
HH
Towndrow
Kelly M
KM
Jia
Zhe
Z
Monks
Terrence J
TJ
Lau
Serrine S
SS
D000595:Amino Acid Sequence; D000818:Animals; D015232:Dinoprostone / Q000031:analogs & derivatives / Q000494:pharmacology; D018374:LLC-PK1 Cells / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis*; D010449:Peptide Mapping / Q000295:instrumentation / Q000379:methods*; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013552:Swine
C481059:11-deoxy,16,16-dimethyl prostaglandin E(2); D010446:Peptide Fragments; D011506:Proteins; D015232:Dinoprostone