Journal of Chromatography A, 1195 (2008) 34–43 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Improving off-line accelerated tryptic digestion Towards fast-lane proteolysis of complex biological samples Jadranka Vukovic a,b ,H˚ avard Loftheim a , Bjørn Winther a , J. L ´ eon E. Reubsaet a,∗ a School of Pharmacy, Department of Pharmaceutical Chemistry, University of Oslo, P.O. Box 1068 Blindern, NO-0316 Oslo, Norway b Faculty of Pharmacy and Biochemistry, Department of Analytics and Control of Medicines, University of Zagreb, Ante Kovaˇ ci´ ca 1, HR-10000 Zagreb, Croatia article info Article history: Received 2 January 2008 Received in revised form 28 March 2008 Accepted 8 May 2008 Available online 13 May 2008 Keywords: Immobilized trypsin BSA Cytochrome c Human urine Proteolysis Digestion characteristics abstract 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 (89min) and elevated temperature (37 ◦ 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 conven- tional in-solution digestion (overnight at 37 ◦ 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. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Proteolysis is an essential step in most proteomic workflows; in general trypsin is used to digest a complex mixture of proteins to yield (or to produce) peptide products which are in its turn iden- tified by tandem MS eventually coupled to chromatography [1,2]. Trypsin cleaves the proteins exclusively at the arginine and lysine residues [3,4] and is traditionally carried out in-solution. Almost 40 years ago the first reports on covalent binding of trypsin to a carrier were published [5]. Because of the currently main focus on proteomics much attention is paid to digestion using immobilized trypsin. For this purpose different carrier materials and formats have been described [6–8] and a thorough review by Ma et al. has been published [9]. From the above-mentioned research, the advantages of digestion using immobilized trypsin are obvious: short reaction time, possible re-use of the enzyme and improved stability. In addition to this, the use of immobilized trypsin allows automation as reviewed by Massolini and Calleri [10]. ∗ Corresponding author. Tel.: +47 22856613; fax: +47 22854402. E-mail address: leonr@farmasi.uio.no (J.L.E. Reubsaet). Experiments carried out by using immobilized trypsin have been focused on the digestion of model proteins like cytochrome c [7], myoglobin [11], bovine serum albumin (BSA) [6], recombinant hemoglobin [12] and sickle cell hemoglobin [13]. Common in these experiments is that these substrates were digested in buffered solu- tions and not in complex biological matrices. These studies showed that digestion could be carried out using immobilized trypsin in various formats. However, only in few experiments protein diges- tion using immobilized trypsin in complex biological samples was used, e.g. the on-chip digestion of rat liver extract and protein iden- tification after separation on a reversed-phase column [14] and the on-line digestion and transthyretin variant identification in human serum [15]. The study described in this paper focuses on the choice of appropriate reactor to perform the accelerated proteolysis using immobilized trypsin in an off-line setup as well as the optimization of the digestion conditions. Comparison with in-solution digests and evaluation of digest efficiency using immobilized trypsin in complex biological samples is performed. This was done using response variables such as visual comparison, signal intensities, sequence coverage, masses matched and amount of missed cleav- ages. 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.05.010