Pepsin immobilized in dextran-modified fused-silica capillaries for on-line protein digestion and peptide mapping.
On-line digestion of proteins under acidic conditions was studied using micro-reactors consisting of dextran-modified fused-silica capillaries with covalently immobilized pepsin. The proteins used in this study differed in molecular weight, isoelectric point and sample composition. The injected protein samples were completely digested in 3 min and the digest was analyzed with micro-high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). The different proteins present in the samples could be identified with a Mascot database search on the basis of auto-MS/MS data. It proved also to be possible to digest and analyze protein mixtures with a sequence coverage of 55% and 97% for the haemoglobin beta- and alpha-chain, respectively, and 35-55% for the various casein variants. Protease auto-digestion, sample carry-over and loss of signal due to adsorption of the injected proteins were not observed. The backpressure of the reactor is low which makes coupling to systems such as Surface Plasmon Resonance biosensors, which do not tolerate too high pressure, possible. The reactor was stable for at least 40 days when used continuously.
Stigter
E C A
EC
Division of Biomedical Analysis, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands. e.c.a.stigter@uu.nl
de Jong
G J
GJ
van Bennekom
W P
WP
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003911:Dextrans / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009862:Online Systems / Q000295:instrumentation*; D010434:Pepsin A / Q000378:metabolism*; D010449:Peptide Mapping / Q000295:instrumentation* / Q000379:methods*; D012822:Silicon Dioxide / Q000737:chemistry*; D013499:Surface Properties; D053719:Tandem Mass Spectrometry
D003911:Dextrans; D004800:Enzymes, Immobilized; D012822:Silicon Dioxide; D010434:Pepsin A