title: A universal SI-traceable isotope dilution mass spectrometry method for protein quantitation in a matrix by tandem mass tag technology.
abstract: Isotope dilution mass spectrometry (IDMS), an important metrological method, is widely used for absolute quantification of peptides and proteins. IDMS employs an isotope-labeled peptide or protein as an internal standard although the use of a protein provides improved accuracy. Generally, the isotope-labeled protein is obtained by stable isotope labeling by amino acids in cell culture (SILAC) technology. However, SILAC is expensive, laborious, and time-consuming. To overcome these drawbacks, a novel universal SI-traceable IDMS method for absolute quantification of proteins in a matrix is described with human transferrin (hTRF). The hTRF and a human serum sample were labeled with different tandem mass tags (TMTs). After mixing the TMT-labeled hTRF and serum sample together followed by digestion, the peptides were separated by nano-liquid chromatography and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Using the signature peptides, we calculated the ratios of reporter ions from the TMT-labeled peptides which, in turn, allowed determination of the mass fraction of hTRF. The recovery ranged from 97% to 105% with a CV of 3.9%. The LOD and LOQ were 1.71 × 10(-5) g/g and 5.69 × 10(-5) g/g of hTRF in human serum, respectively, and the relative expanded uncertainty was 4.7% with a mass fraction of 2.08 mg/g. For comparison, an enzyme-linked immunosorbent assay (ELISA) method for hTRF yielded a mass fraction of 2.03 mg/g. This method provides a starting point for establishing IDMS technology to accurately determine the mass fractions of protein biomarkers in a matrix with traceability to SI units. This technology should support the development of a metrological method useful for quantification of a wide variety of proteins.
authors.affiliation: College of Science, Beijing University of Chemical Technology, No.15, Beisanhuan East Road, Chaoyang District, Beijing, China.
authors.forename: Jiale
authors.initials: J
authors.lastname: Li
authors.affiliation: Division of Medical and Biological Measurements, National Institute of Metrology, Beijing, China.
authors.forename: Liqing
authors.initials: L
authors.lastname: Wu
authors.affiliation: Division of Medical and Biological Measurements, National Institute of Metrology, Beijing, China.
authors.forename: Youxun
authors.initials: Y
authors.lastname: Jin
authors.affiliation: College of Science, Beijing University of Chemical Technology, No.15, Beisanhuan East Road, Chaoyang District, Beijing, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Su
authors.affiliation: Division of Medical and Biological Measurements, National Institute of Metrology, Beijing, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Yang
authors.affiliation: College of Science, Beijing University of Chemical Technology, No.15, Beisanhuan East Road, Chaoyang District, Beijing, China. yangyi@mail.buct.edu.cn.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007553:Isotope Labeling; D057230:Limit of Detection; D010455:Peptides / Q000097:blood; D012825:Silicon / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014168:Transferrin / Q000032:analysis* / Q000737:chemistry
chemical_list: D010455:Peptides; D014168:Transferrin; D012825:Silicon
keywords: Isotope dilution mass spectrometry method; Protein quantitation; SI-traceable; Tandem mass tag technology