Elucidation of the primary structures of proteins by mass spectrometry.
A combination of new mass spectrometric methods that can be used to determine the primary structures of proteins, including post-translational modifications, with unprecedented speed and accuracy is described. Structural characterization of alpha-crystallins from bovine lenses has been used to illustrate the methods. The molecular weights of alpha-crystallins fractionated, but not to homogeneity, by reversed-phase HPLC were determined with an uncertainty of 0.01% which is at least 100 times more accurate than is possible using conventional methods. This information was used to identify the primary gene product as well as its phosphorylated and truncated forms. Molecular weight maps of proteolytic digests of these proteins were determined by directly coupled capillary HPLC fast atom bombardment-mass spectrometry. From these maps, the entire amino acid sequence was confirmed, and the phosphorylated peptide was identified. The MS/MS daughter ion mass spectrum of the phosphorylated peptide provided sufficient information to determine which residue was phosphorylated. Because protein structure, including post-translational modifications, is determined on the basis of molecular weight, this method has broad application and will be useful for a variety of diverse and challenging problems in protein structure elucidation.
Smith
J B
JB
Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907.
Thévenon-Emeric
G
G
Smith
D L
DL
Green
B
B
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003459:Crystallins / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D016339:Spectrometry, Mass, Fast Atom Bombardment
D003459:Crystallins