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Fast protein sequence determination with matrix‐assisted laser desorption and ionization mass spectrometry
Author(s) -
Schär Martin,
Börnsen K. Olaf,
Gassmann Ernst
Publication year - 1991
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.1290050705
Subject(s) - chemistry , mass spectrometry , chromatography , protein mass spectrometry , sample preparation in mass spectrometry , amino acid , molecular mass , peptide sequence , matrix assisted laser desorption/ionization , peptide , bottom up proteomics , matrix (chemical analysis) , carboxypeptidase , proteolytic enzymes , biochemistry , desorption , enzyme , tandem mass spectrometry , electrospray ionization , organic chemistry , adsorption , gene
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 molecular mass of 4117.8 Da was digested with carboxypeptidases Y and B and the seqence 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 trypitc 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 norminal 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 digestin time due to high enzyme/substrate rations, 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.