title: The pros and cons of increased trypsin-to-protein ratio in targeted protein analysis.
abstract: The effect of increasing the trypsin amount in bottom-up based targeted protein analysis is evaluated. By applying an increased trypsin-to-protein ratio (1:1 (w/w)) after heat denaturation (60 °C), reduction and alkylation, the digestion time could be reduced profoundly compared to conventional digestion conditions (ratio 1:40, overnight) without compromising method sensitivity or digestion repeatability. The procedure was obtained after a systematic evaluation of trypsin level and trypsin quality using a set of three model proteins: human chorionic gonadotropin (hCG), bovine serum albumin (BSA) and cytochrome C (CytC). All peptides monitored were produced at similar or higher levels after 45 min at trypsin-to-protein ratio 1:1, compared to conventional overnight digestion (exception: CytC using modified trypsin, required up to 4 h (at 1:1 ratio) in order achieve this). Peptide decay due to chymotryptic activity was observed at longer digestion times, but the effect was circumvented using digestion times <4 h. The accelerated digestion protocol (1:1 (w/w), 45 min) was applied to realistic human serum samples containing the biomarker protein hCG to demonstrate its applicability.
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, Oslo, Norway.
authors.forename: Siri Valen
authors.initials: SV
authors.lastname: Egeland
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, Oslo, Norway.
authors.forename: Léon
authors.initials: L
authors.lastname: Reubsaet
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, Oslo, Norway. Electronic address: trinegha@farmasi.uio.no.
authors.forename: Trine Grønhaug
authors.initials: TG
authors.lastname: Halvorsen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002417:Cattle; D006063:Chorionic Gonadotropin / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism*
chemical_list: D006063:Chorionic Gonadotropin; D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; D012710:Serum Albumin, Bovine; D045304:Cytochromes c; D014357:Trypsin
keywords: Accelerated digestion; Bottom-up proteomics; LC-MS/MS; Peptide decay; Targeted protein analysis; Trypsin