Characterization of specific noncovalent protein complexes by UV matrix-assisted laser desorption ionization mass spectrometry.
While electrospray (ESI) mass spectrometry has already established its potential for the characterization of non-covalent protein complexes, matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) seemed not to be applicable hitherto because of limitations in matrix chemistry and sample preparation. In this work, a sample preparation method has been developed in which 6-aza-2-thiothymine (ATT) was used as a matrix without any addition of organic cosolvents, and proteins were dissolved in aqueous buffers such as ammonium hydrogencarbonate, ammonium citrate and ammonium acetate. Under these conditions, the intact non-covalent protein complexes, RNAse S, the non-covalent complex of S-protein and S-peptide and specific dimers of coiled-coil leucine zipper polypeptides were observed by UV-MALDI/MS. The specificity of complex formation was ascertained by admixture of non-specific peptides which did not yield detectable aggregate ions. In addition, on-target tryptic digestion of cytochrome c and leucine zipper peptides was carried out after MALDI/MS molecular mass determination in the presence of the ATT matrix. Mass spectrometric analyses of these tryptic digests yielded spectra that showed complete digestion of the proteins. These results indicate that proteins maintained intact tertiary structures necessary for the formation of specific non-covalent complexes, and that trypsin retained its functional enzymatic structure and full biological activity with the present sample preparation method.
Glocker
M O
MO
Faculty of Chemistry, University of Konstanz, Germany.
Bauer
S H
SH
Kast
J
J
Volz
J
J
Przybylski
M
M
D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D006868:Hydrolysis; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D012260:Ribonucleases / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin
D010455:Peptides; D011506:Proteins; D012260:Ribonucleases; D014357:Trypsin