Chemically modified, immobilized trypsin reactor with improved digestion efficiency.
Tryptic digestion followed by identification using mass spectrometry is an important step in many proteomic studies. Here, we describe the preparation of immobilized, acetylated trypsin for enhanced digestion efficacy in integrated protein analysis platforms. Complete digestion of cytochrome c was obtained with two types of modified-trypsin beads with a contact time of only 4 s, while corresponding unmodified-trypsin beads gave only incomplete digestion. The digestion rate of myoglobin, a protein known to be rather resistant to proteolysis, was not altered by acetylating trypsin and required a buffer containing 35% acetonitrile to obtain complete digestion. The use of acetylated-trypsin beads led to fewer interfering tryptic autolysis products, indicating an increased stability of this modified enzyme. Importantly, the modification did not affect trypsin's substrate specificity, as the peptide map of myoglobin was not altered upon acetylation of immobilized trypsin. Kinetic digestion experiments in solution with low-molecular-weight substrates and cytochrome c confirmed the increased catalytic efficiency (lower K(M) and higher k(cat)) and increased resistance to autolysis of trypsin upon acetylation. Enhancement of catalytic efficiency was correlated with the number of acetylations per molecule. The favorable properties of the new chemically modified trypsin reactor should make it a valuable tool in automated protein analysis systems.
Freije
J Robert
JR
Center for Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Mulder
Patty P M F A
PP
Werkman
Wendy
W
Rieux
Laurent
L
Niederlander
Harm A G
HA
Verpoorte
Elisabeth
E
Bischoff
Rainer
R
D000097:Acetonitriles / Q000737:chemistry / Q000494:pharmacology; D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D045304:Cytochromes c / Q000737:chemistry*; D006736:Horses; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry; D010449:Peptide Mapping; D011506:Proteins / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology*
D000097:Acetonitriles; D009211:Myoglobin; D011506:Proteins; D020543:Proteome; D045304:Cytochromes c; D014357:Trypsin; C032159:acetonitrile