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High sequence coverage by in‐capillary proteolysis of native proteins and simultaneous analysis of the resulting peptides by nanoelectrospray ionization‐mass spectrometry and tandem mass spectrometry
Author(s) -
Pohlentz Gottfried,
Kölbl Stefanie,
PeterKatalinić Jasna
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401060
Subject(s) - chemistry , chromatography , trypsin , bottom up proteomics , mass spectrometry , tandem mass spectrometry , peptide , proteolysis , capillary electrophoresis , peptide mass fingerprinting , casein , electrospray ionization , electrospray , protein mass spectrometry , biochemistry , proteomics , enzyme , gene
Losses of proteolytic peptides during extraction and/or purification procedures succeeding in‐gel or in‐solution digests of proteins frequently occur in the course of protein identification investigations. In order to overcome this disadvantage, the method of in‐capillary digest was developed: native proteins were incubated in the presence of endoproteases in the electrospray capillary and the resulting peptides were analyzed by nanoelectrospray‐mass spectrometry during the ongoing proteolysis. In‐capillary digest of apomyglobin by use of trypsin in a molar ratio of 25:1 yielded complete degradation already after 15 min. The sequence coverage based on formation of molecular ions was 100% and peptide ions could be fragmented by collision‐induced dissociation and sequenced. When myoglobin was incubated in the electrospray capillary with trypsin in a molar ratio of 500:1, a clear shift from molecular ions and miscleaved peptide ions to the expected final tryptic peptide ions was observed over a 2 h period. The peptide spectra obtained from tryptic in‐capillary proteolysis of bovine serum albumin and apotransferrin, respectively, gave rise to sequence coverages of more than 40% for both proteins. The data obtained from the peptide maps as well as from collision‐induced dissociation (CID) of selected peptides were more than sufficient for protein identification by database searches. An elephant milk protein preparation was used to demonstrate the application of in‐capillary proteolysis on protein mixtures. Tryptic digest, simultaneous analysis of the proteolytic peptides by use of CID, and subsequent sequencing allowed the identification of lactoferrin, αs1‐casein, β‐casein, δ‐casein, and κ‐casein by homology search.