title: Use of stable isotope dimethyl labeling coupled to selected reaction monitoring to enhance throughput by multiplexing relative quantitation of targeted proteins.
abstract: In this manuscript, we present a proof-of-concept study for targeted relative protein quantitation workflow using chemical labeling in the form of dimethylation, coupled with selected reaction monitoring (dimethyl-SRM). We first demonstrate close to complete isotope incorporation for all peptides tested. The accuracy, reproducibility, and linear dynamic range of quantitation are further assessed based on known ratios of nonhuman standard proteins spiked into human cerebrospinal fluid (CSF) as a model complex matrix. Quantitation reproducibility below 20% (CV < 20%) was obtained for analyte concentrations present at a dynamic range of 4 orders of magnitude lower than that of the background proteins. An error of less than 15% was observed when measuring the abundance of 44 out of 45 major human plasma proteins. Dimethyl-SRM was further examined by comparing the relative quantitation of eight proteins in human CSF with the relative quantitation obtained using synthetic heavy peptides coupled to stable isotope dilution-SRM (SID-SRM). Comparison between the two methods reveals that the correlation between dimethyl-SRM and SID-SRM is within 0.3-33% variation, demonstrating the accuracy of relative quantitation using dimethyl-SRM. Dimethyl labeling coupled with SRM provides a fast, convenient, and cost-effective alternative for relative quantitation of a large number of candidate proteins/peptides.
authors.affiliation: Proteomics Unit, Department of Biomedicine, University of Bergen, Bergen, Norway. Thin.aye@biomed.uib.no
authors.forename: Thin Thin
authors.initials: TT
authors.lastname: Aye
authors.forename: Teck Yew
authors.initials: TY
authors.lastname: Low
authors.forename: Yngvild
authors.initials: Y
authors.lastname: Bjørlykke
authors.forename: Harald
authors.initials: H
authors.lastname: Barsnes
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
authors.forename: Frode S
authors.initials: FS
authors.lastname: Berven
mesh_terms: D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000032:analysis*; D006801:Humans; D007201:Indicator Dilution Techniques; D007553:Isotope Labeling / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000134:cerebrospinal fluid*; D040901:Proteomics / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results
chemical_list: D001798:Blood Proteins; D010455:Peptides