C-Terminal Sequence Analysis of Peptides and Proteins Using Carboxypeptidases and Mass Spectrometry after Derivatization of Lys and Cys Residues

Valentina Bonetto, Ann Charlotte Bergman, Hans Jörnvall, Rannar Sillard

Research output: Contribution to journalArticle

Abstract

C-Terminal sequence analysis of peptides and proteins using carboxypeptidase digestion in combination with matrix-assisted laser desorption/ionization mass spectrometxy (MALDI-MS) is convenient for protein and peptide characterization. After a short digestion, a sequence up to 20 residues can be identified, but the total number depends on the individual sequence. Due to the accuracy limits of the MALDI time-of-flight arrangement, the assignment of several residues with close mass values, including Lys/Glx, may remain ambiguous. We have used derivatization of lysine residues by guanidination to overcome the problem of Lys identification. The reaction is rapid and specific and results in full derivatization. In the case of Cys-containing peptides, problems arise from the fact that carboxypeptidases Y and P do not cleave peptides that contain nonderivatized cystine, cysteic acid, or (carboxymethyl)cysteine. Successful identification of Cys residues within the sequence is instead achieved by conversion of Cys to 4-thialaminine by (trimethyiamino)ethylation. The two derivatizations of Lys and Cys side chains provide opportunities for proton attachment and therefore facilitate the analysis by MALDI-MS. This C-terminal sequence analysis method is also useful for large proteins after fragmentation with specific enzymes.

Original languageEnglish
Pages (from-to)1315-1319
Number of pages5
JournalAnalytical Chemistry
Volume69
Issue number7
Publication statusPublished - 1997

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Carboxypeptidases
Mass spectrometry
Peptides
Ionization
Desorption
Proteins
Cysteic Acid
Cathepsin A
Cystine
Lasers
Lysine
Cysteine
Protons
Enzymes

ASJC Scopus subject areas

  • Analytical Chemistry

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C-Terminal Sequence Analysis of Peptides and Proteins Using Carboxypeptidases and Mass Spectrometry after Derivatization of Lys and Cys Residues. / Bonetto, Valentina; Bergman, Ann Charlotte; Jörnvall, Hans; Sillard, Rannar.

In: Analytical Chemistry, Vol. 69, No. 7, 1997, p. 1315-1319.

Research output: Contribution to journalArticle

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