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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

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.

Graphical Abstract

Absolute quantification of hTRF in human serum by TMT-IDMS

Keywords

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

Abbreviations

ACN

Acetonitrile

CHCA

(R)-cyano-4-hydroxycinnamic acid

CID

Collision-induced dissociation

CRM

Certified reference material

CV

Coefficient of variation

ELISA

Enzyme-linked immunosorbent assay

HSA

Human serum albumin

hGH

Human growth hormone

hCG

Human chorionic gonadotropin

hTRF

Human transferrin

ICP-MS

Inductively coupled plasma mass spectrometry

IDMS

Isotope dilution mass spectrometry

LOD

Limit of detection

LOQ

Limit of quantification

MALDI-TOF-MS

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

SA

Sinapinic acid

SDS

Sodium dodecyl sulfate

SILAC

Stable isotope labeling by amino acids in cell culture

TEAB

Triethylammonium bicarbonate

TFA

Trifluoroacetic acid

TMTs

Tandem mass tags

Notes

Acknowledgments

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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