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

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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.

Absolute quantification of hTRF in human serum by TMT-IDMS

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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

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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).

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Authors and Affiliations

  1. College of Science, Beijing University of Chemical Technology, No.15, Beisanhuan East Road, Chaoyang District, Beijing, China

    Jiale Li, Ping Su & Yi Yang

  2. Division of Medical and Biological Measurements, National Institute of Metrology, Beijing, China

    Liqing Wu, Youxun Jin & Bin Yang

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  1. Jiale Li
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Correspondence to Yi Yang.

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

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Li, J., Wu, L., Jin, Y. et al. A universal SI-traceable isotope dilution mass spectrometry method for protein quantitation in a matrix by tandem mass tag technology. Anal Bioanal Chem 408, 3485–3493 (2016). https://doi.org/10.1007/s00216-016-9424-0

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