title: An improved trypsin digestion method minimizes digestion-induced modifications on proteins.
abstract: Trypsin digestion can induce artificial modifications such as asparagine deamidation and N-terminal glutamine cyclization on proteins due to the temperature and the alkaline pH buffers used during digestion. The amount of these artificial modifications is directly proportional to the incubation time of protein samples in the reduction/alkylation buffer and, more important, in the digestion buffer where the peptides are completely solvent exposed. To minimize these artificial modifications, we focused on minimizing the trypsin digestion time by maximizing trypsin activity. Trypsin activity was optimized by the complete removal of guanidine, which is a known trypsin inhibitor, from the digestion buffer. As a result, near complete trypsin digestion was achieved on reduced and alkylated immunoglobulin gamma molecules in 30min. The protein tryptic fragments and their modification products were analyzed and quantified by reversed-phase liquid chromatography/tandem mass spectrometry using an in-line LTQ Orbitrap mass spectrometer. The reduction and alkylation reaction time was also minimized by monitoring the completeness of the reaction using a high-resolution time-of-flight mass spectrometer. Using this 30-min in-solution trypsin digestion method, little protocol-induced deamidation or N-terminal glutamine cyclization product was observed and cleaner tryptic maps were obtained due to less trypsin self-digestion and fewer nonspecific cleavages. The throughput of trypsin digestion was also improved significantly compared with conventional trypsin digestion methods.
authors.affiliation: Department of Formulation and Analytical Resources, Amgen, Thousand Oaks, CA 91320, USA. dren@amgen.com
authors.forename: Da
authors.initials: D
authors.lastname: Ren
authors.forename: Gary D
authors.initials: GD
authors.lastname: Pipes
authors.forename: Dingjiang
authors.initials: D
authors.lastname: Liu
authors.forename: Liang-Yu
authors.initials: LY
authors.lastname: Shih
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Nichols
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Treuheit
authors.forename: David N
authors.initials: DN
authors.lastname: Brems
authors.forename: Pavel V
authors.initials: PV
authors.lastname: Bondarenko
mesh_terms: D000478:Alkylation; D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000379:methods*; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism*
chemical_list: D007074:Immunoglobulin G; D014357:Trypsin