Analytical and Bioanalytical Chemistry

, Volume 408, Issue 13, pp 3485–3493 | Cite as

A universal SI-traceable isotope dilution mass spectrometry method for protein quantitation in a matrix by tandem mass tag technology

  • Jiale Li
  • Liqing Wu
  • Youxun Jin
  • Ping Su
  • Bin Yang
  • Yi YangEmail author
Research Paper


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.

Graphical Abstract

Absolute quantification of hTRF in human serum by TMT-IDMS


SI-traceable Isotope dilution mass spectrometry method Protein quantitation Tandem mass tag technology 





(R)-cyano-4-hydroxycinnamic acid


Collision-induced dissociation


Certified reference material


Coefficient of variation


Enzyme-linked immunosorbent assay


Human serum albumin


Human growth hormone


Human chorionic gonadotropin


Human transferrin


Inductively coupled plasma mass spectrometry


Isotope dilution mass spectrometry


Limit of detection


Limit of quantification


Matrix-assisted laser desorption ionization time-of-flight mass spectrometry


Sinapinic acid


Sodium dodecyl sulfate


Stable isotope labeling by amino acids in cell culture


Triethylammonium bicarbonate


Trifluoroacetic acid


Tandem mass tags



This work is part of the project funded by the Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China (AQSIQ, grant no. 201310008) and National Institute of Metrology, China (NIM, grant no. AKY1413).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical consent

Human serum samples were collected from the healthy volunteers after taking their written consent and the approval by the ethical committee of the institute.

Supplementary material

216_2016_9424_MOESM1_ESM.pdf (234 kb)
ESM 1 (PDF 234 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Jiale Li
    • 1
  • Liqing Wu
    • 2
  • Youxun Jin
    • 2
  • Ping Su
    • 1
  • Bin Yang
    • 2
  • Yi Yang
    • 1
    Email author
  1. 1.College of ScienceBeijing University of Chemical TechnologyBeijingChina
  2. 2.Division of Medical and Biological MeasurementsNational Institute of MetrologyBeijingChina

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