A universal SI-traceable isotope dilution mass spectrometry method for protein quantitation in a matrix by tandem mass tag technology
| Autor: | Bin Yang, Yi Yang, Liqing Wu, Ping Su, Li Jiale, Jin Youxun |
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| Rok vydání: | 2015 |
| Předmět: |
0301 basic medicine
Silicon Protein mass spectrometry Enzyme-Linked Immunosorbent Assay Isotope dilution Tandem mass spectrometry Mass spectrometry Tandem mass tag 01 natural sciences Biochemistry Sample preparation in mass spectrometry Analytical Chemistry Matrix (chemical analysis) 03 medical and health sciences Limit of Detection Tandem Mass Spectrometry Stable isotope labeling by amino acids in cell culture Humans Amino Acid Sequence Chromatography Chemistry 010401 analytical chemistry Transferrin 0104 chemical sciences 030104 developmental biology Isotope Labeling Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Peptides Chromatography Liquid |
| Zdroj: | Analytical and bioanalytical chemistry. 408(13) |
| ISSN: | 1618-2650 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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