Improving off-line accelerated tryptic digestion. Towards fast-lane proteolysis of complex biological samples.
Off-line digestion of proteins using immobilized trypsin beads is studied with respect to the format of the digestion reactor, the digestion conditions, the comparison with in-solution digestion and its use in complex biological samples. The use of the filter vial as the most appropriate digestion reactor enables simple, efficient and easy-to-handle off-line digestion of the proteins on trypsin beads. It was shown that complex proteins like bovine serum albumin (BSA) need much longer time (89 min) and elevated temperature (37 degrees C) to be digested to an acceptable level compared to smaller proteins like cytochrome c (5 min, room temperature). Comparing the BSA digestion using immobilized trypsin beads with conventional in-solution digestion (overnight at 37 degrees C), it was shown that comparable results were obtained with respect to sequence coverage (>90%) and amount of missed cleavages (in both cases around 20 peptides with 1 or 2 missed cleavages were detected). However, the digestion using immobilized trypsin beads was considerable less time consuming. Good reproducibility and signal intensities were obtained for the digestion products of BSA in a complex urine sample. In addition to this, peptide products of proteins typically present in urine were identified.
Vukovic
Jadranka
J
School of Pharmacy, Department of Pharmaceutical Chemistry, University of Oslo, P.O. Box 1068 Blindern, NO-0316 Oslo, Norway.
Loftheim
Håvard
H
Winther
Bjørn
B
Reubsaet
J Léon E
JL
D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D045304:Cytochromes c / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D012710:Serum Albumin, Bovine / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D013696:Temperature; D014357:Trypsin / Q000378:metabolism*
D004800:Enzymes, Immobilized; D012710:Serum Albumin, Bovine; D045304:Cytochromes c; D014357:Trypsin