title: Optimization of digestion parameters for protein quantification.
abstract: We present a rapid and efficient in-solution enzymatic digestion protocol suitable for mass spectrometry-based absolute protein quantification techniques. The digestion method employs RapiGest SF (an acid-labile surfactant), an excess amount of modified trypsin (enzyme-to-substrate ratio of 2.5:1), and an incubation time of 2 h. No reduction/alkylation reagents are used. Digestion parameters were varied systematically to monitor their effect on rate and completeness of digestion. To demonstrate the general applicability of the method, the optimization was done using a viral hemagglutinin (HA) as a model protein and then applied to ricin, a potent protein toxin extracted from the castor bean (Ricinus communis). The parameters that were optimized included incubation time, concentration of RapiGest SF, enzyme-to-substrate ratio, and incubation temperature. The optimization was done by comparing the yields from two protein-specific peptides originating from two different sites of the HA protein. The analysis was performed by liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode using isotopically labeled peptide standards for quantification.
authors.affiliation: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway, MS F-50, Atlanta, GA 30341, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Norrgran
authors.forename: Tracie L
authors.initials: TL
authors.lastname: Williams
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Woolfitt
authors.forename: Maria I
authors.initials: MI
authors.lastname: Solano
authors.forename: James L
authors.initials: JL
authors.lastname: Pirkle
authors.forename: John R
authors.initials: JR
authors.lastname: Barr
mesh_terms: D000595:Amino Acid Sequence; D001671:Biochemistry / Q000379:methods*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D053124:Influenza A Virus, H5N1 Subtype / Q000378:metabolism*; D008969:Molecular Sequence Data; D012276:Ricin / Q000032:analysis / Q000378:metabolism*; D012278:Ricinus / Q000737:chemistry
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D012276:Ricin