Fast protein sequence determination with matrix-assisted laser desorption and ionization mass spectrometry.
Measurement and identification of digested peptides by matrix-assisted laser desorption and ionization mass spectrometry (LDI-MS) is demonstrated. Synthetic human parathyroid hormone, pTH (1-34), with a molecular mass of 4117.8 Da was digested with carboxypeptidases Y and B and the sequence of 14 amino acids from the C-terminus of the peptide was determined by analyzing the molecular mass of the truncated peptides. Furthermore, a tryptic digestion of pTH (1-34) was carried out and a molecular mass map of pTH (1-34) was obtained. With the results of the proteolytic digestion a rapid confirmation of the amino-acid sequence of the protein was possible. It is shown that the results of the tryptic digestion can be used for the unambiguous identification of the amino acid residues Lys and Arg, which cannot be distinguished with a mass spectrometer because of their equal nominal masses. Several advantages of amino acid sequence determination by the combination of digestion and LDI-MS are obvious: high sensitivity in the low pmol range, fast digestion time due to high enzyme/substrate ratios, quantification is unnecessary because the amino acids are identified by their molecular mass differences, the low chemical expenditure for the digestions and the accuracy of the sequence determination. Measurements with LDI-MS are fast: sample preparation and the measurement take only a few min. The mass determination and amino acid sequence is completely unimpaired by amino acid contaminations or impurities in the sample. The sensitivity of the method is in the low pmol to fmol range and thus comparable to other analytical methods.
Schär
M
M
Central Analytical Department, Ciba-Geigy Ltd., Basel, Switzerland.
Börnsen
K O
KO
Gassmann
E
E
D000595:Amino Acid Sequence; D002268:Carboxypeptidases; D003373:Coumaric Acids / Q000032:analysis; D006801:Humans; D006868:Hydrolysis; D007834:Lasers; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000032:analysis; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D019379:Teriparatide; D014357:Trypsin
D003373:Coumaric Acids; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D010455:Peptides; D019379:Teriparatide; C073734:sinapinic acid; D002268:Carboxypeptidases; D014357:Trypsin