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.
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, alphas1-casein, beta-casein, delta-casein, and kappa-casein by homology search.
Pohlentz
Gottfried
G
Institute for Medical Physics and Biophysics, University of Münster, Germany. pohlentz@uni-muenster.de
Kölbl
Stefanie
S
Peter-Katalinić
Jasna
J
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004606:Elephants; D005260:Female; D008894:Milk Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014168:Transferrin / Q000737:chemistry / Q000235:genetics; D014357:Trypsin
D001059:Apoproteins; D008894:Milk Proteins; D009211:Myoglobin; D010446:Peptide Fragments; D014168:Transferrin; C010857:apomyoglobin; C028284:apotransferrin; D012710:Serum Albumin, Bovine; D014357:Trypsin