title: A new strategy of using O18-labeled iodoacetic acid for mass spectrometry-based protein quantitation.
abstract: A new O(18) labeling protocol is designed to assist quantitation of cysteine-containing proteins using LC/MS. Unlike other O(18) labeling strategies, the labeling is carried out at the intact protein level (prior to its digestion) during reduction/alkylation of cysteine side chains using O(18)-labeled iodoacetic acid (IAA). The latter can be easily prepared by exchanging carboxylic oxygen atoms of commercially available IAA in O(18)-enriched water at low pH. Since incorporation of the O(18) label in the protein occurs at the whole protein, rather than peptide level, the quantitation results are not peptide-dependent. The excellent stability of the label in mild pH conditions provides flexibility and robustness needed of sample processing steps following the labeling. In contrast to generally costly isotope labeling reagents, this approach uses only two relatively inexpensive commercially available reagents (IAA and H(2)O(18)). The feasibility of the new method is demonstrated using an 80 kDa human serum transferrin (hTf) as a model, where linear quantitation is achieved across a dynamic range spanning three orders of magnitude. The new approach can be used in quantitative proteomics applications and is particularly suitable for a variety of tasks in the biopharmaceutical sector, ranging from pharmacokinetic studies to quality control of protein therapeutics.
authors.affiliation: Department of Chemistry, University of Massachusetts Amherst, 710 North Pleasant Street, LGRT 104, Amherst, MA 01003, USA.
authors.forename: Shunhai
authors.initials: S
authors.lastname: Wang
authors.forename: Igor A
authors.initials: IA
authors.lastname: Kaltashov
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D003545:Cysteine / Q000032:analysis / Q000737:chemistry; D005240:Feasibility Studies; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019807:Iodoacetic Acid / Q000737:chemistry*; D007553:Isotope Labeling / Q000379:methods*; D008969:Molecular Sequence Data; D010103:Oxygen Isotopes / Q000737:chemistry*; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014168:Transferrin / Q000032:analysis / Q000737:chemistry
chemical_list: D010103:Oxygen Isotopes; D010446:Peptide Fragments; D011506:Proteins; D014168:Transferrin; D003545:Cysteine; D019807:Iodoacetic Acid