Immobilized trypsin systems coupled on-line to separation methods: recent developments and analytical applications.
The ability to rapidly and efficiently digest and identify an unknown protein is of great utility for proteome studies. Identification of proteins via peptide mapping is generally accomplished through proteolytic digestion with enzymes such as trypsin. Limitations of this approach consist in manual sample manipulation steps and extended reaction times for proteolytic digestion. The use of immobilized trypsin for cleavage of proteins is advantageous in comparison with application of its soluble form. Enzymes can be immobilized on different supports and used in flow systems such as immobilized enzyme reactors (IMERs). This review reports applications of immobilized trypsin reactors in which the IMER has been integrated into separation systems such as reversed-phase liquid chromatography or capillary electrophoresis, prior to MS analysis. Immobilization procedures including supports, mode of integration into separation systems, and methods are described.
Massolini
Gabriella
G
Department of Pharmaceutical Chemistry, University of Pavia, Via Taramelli 12, 27100 Pavia, Italy. g.massolini@unipv.it
Calleri
Enrica
E
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods; D004800:Enzymes, Immobilized; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping / Q000295:instrumentation / Q000379:methods; D011108:Polymers; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D012822:Silicon Dioxide; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods; D014357:Trypsin
D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D011108:Polymers; D011506:Proteins; D012822:Silicon Dioxide; D014357:Trypsin