title: Critical assessment of accelerating trypsination methods.
abstract: In LC-MS based proteomics, several accelerating trypsination methods have been introduced in order to speed up the protein digestion, which is often considered a bottleneck. Traditionally and most commonly, due to sample heterogeneity, overnight digestion at 37 °C is performed in order to digest both easily and more resistant proteins. High efficiency protein identification is important in proteomics, hours with LC-MS/MS analysis is needless if the majority of the proteins are not digested. Based on preliminary experiments utilizing some of the suggested accelerating methods, the question of whether accelerating digestion methods really provide the same protein identification efficiency as the overnight digestion was asked. In the present study we have evaluated four different accelerating trypsination methods (infrared (IR) and microwave assisted, solvent aided and immobilized trypsination). The methods were compared with conventional digestion at 37 °C in the same time range using a four protein mixture. Sequence coverage and peak area of intact proteins were used for the comparison. The accelerating methods were able to digest the proteins, but none of the methods appeared to be more efficient than the conventional digestion method at 37 °C. The conventional method at 37 °C is easy to perform using commercially available instrumentation and appears to be the digestion method to use. The digestion time in targeted proteomics can be optimized for each protein, while in comprehensive proteomics the digestion time should be extended due to sample heterogeneity and influence of other proteins present. Recommendations regarding optimizing and evaluating the tryptic digestion for both targeted and comprehensive proteomics are given, and a digestion method suitable as the first method for newcomers in comprehensive proteomics is suggested.
authors.affiliation: Department of Chemistry, University of Oslo, PO Box 1033, Blindern, NO-0315 Oslo, Norway.
authors.forename: Hanne Kolsrud
authors.initials: HK
authors.lastname: Hustoft
authors.forename: Leon
authors.initials: L
authors.lastname: Reubsaet
authors.forename: Tyge
authors.initials: T
authors.lastname: Greibrokk
authors.forename: Elsa
authors.initials: E
authors.lastname: Lundanes
authors.forename: Helle
authors.initials: H
authors.lastname: Malerod
mesh_terms: D000478:Alkylation; D002853:Chromatography, Liquid; D006358:Hot Temperature; D008872:Microwaves; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000379:methods*; D011489:Protein Denaturation; D040901:Proteomics; D012997:Solvents; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry*
chemical_list: D012997:Solvents; D014357:Trypsin