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. 2011 Mar 30;6(3):e17282.
doi: 10.1371/journal.pone.0017282.

Quantitative mass spectrometry evaluation of human retinol binding protein 4 and related variants

Urban A Kiernan  1 , David A Phillips, Olgica Trenchevska, Dobrin Nedelkov
Affiliations

Quantitative mass spectrometry evaluation of human retinol binding protein 4 and related variants

Urban A Kiernan et al. PLoS One. .

Abstract

Background: Retinol Binding Protein 4 (RBP4) is an exciting new biomarker for the determination of insulin resistance and type 2 diabetes. It is known that circulating RBP4 resides in multiple variants which may provide enhanced clinical utility, but conventional immunoassay methods are blind to such differences. A Mass Spectrometric immunoassay (MSIA) technology that can quantitate total RBP4 as well as individual isoforms may provide an enhanced analysis for this biomarker.

Methods: RBP4 was isolated and detected from 0.5 uL of human plasma using MSIA technology, for the simultaneous quantification and differentiation of endogenous human RBP4 and its variants.

Results: The linear range of the assay was 7.81-500 ug/mL, and the limit of detection and limit of quantification were 3.36 ug/mL and 6.52 ug/mL, respectively. The intra-assay CVs were determined to be 5.1% and the inter-assay CVs were 9.6%. The percent recovery of the RBP4-MSIA ranged from 95 - 105%. Method comparison of the RBP4 MSIA vs the Immun Diagnostik ELISA yielded a Passing & Bablok fit of MSIA = 1.05× ELISA - 3.09, while the Cusum linearity p-value was >0.1 and the mean bias determined by the Altman Bland test was 1.2%.

Conclusion: The novel RBP4 MSIA provided a fast, accurate and precise quantitative protein measurement as compared to the standard commercially available ELISA. Moreover, this method also allowed for the detection of RBP4 variants that are present in each sample, which may in the future provide a new dimension in the clinical utility of this biomarker.

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Conflict of interest statement

Competing Interests: 1) The authors own stock in the institution they are affiliated with, 2) the authors are employed by the affiliated institution and were paid to perform the research presented, and 3) this research was funded by a research grant provided by the NIH. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Describes the MSIA workflow and provides examples of the normalized quantitative MS responses.
A) Work flow for the quantitative analysis of RBP4 from human plasma by MSIA. B) Selected quantitative MS responses of the RBP4 MSIA.
Figure 2
Figure 2. Shows plots of RBP4 control and calibrator samples analyzed by MSIA.
Samples ranged in concentration from 7.06 to 500 ug/mL (0.30 to 24.31 nmol/mL) and the data represents the results obtained over an eleven day period.
Figure 3
Figure 3. Are the plotted results of the quantitative MSIA linearity study.
The regression analysis was only able to fit a first order polynomial (1.087+3.1016×; R2 = 0.990) to the data set. The absence of non-linearity describes the assay as possessing linear characteristics.
Figure 4
Figure 4. Is a comparison between quantitative MSIA and the Immun Diagnostik ELISA for RBP4.
A) RBP4 concentrations (ug/mL) obtained by both methods were compared by same-scale Passing & Bablok regression (solid black line). The dashed lines are the 95% confidence interval. B) The Altman-Bland test determined that the overall bias of the MSIA to be 1.195 (solid black line). The dashed lines represent the 95% limit of agreement.
Figure 5
Figure 5. Shows representative MS traces of RBP4 acquired during the method comparison study.
The normalized mass spectra display a wide variety of RBP4 variation found in human plasma.
See this image and copyright information in PMC

References

    1. Anderson NL, Anderson NG, Pearson TW, Borchers CH, Paulovich AG, et al. A human proteome detection and quantitation project. Mol Cell Proteomics. 2009;8:883–886. - PMC - PubMed
    1. Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry. 1971;8:871–874. - PubMed
    1. Van Weemen BK, Schuurs AH. Immunoassay using antigen-enzyme conjugates. FEBS Lett. 1971;15:232–236. - PubMed
    1. Anderson NL, Anderson NG, Haines LR, Hardie DB, Olafson RW, et al. Mass spectrometric quantitation of peptides and proteins using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA). J Proteome Res. 2004;3:235–244. - PubMed
    1. Lopez MF, Rezai T, Sarracino DA, Prakash A, Krastins B, et al. Selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants. Clin Chem. 2010;56:281–290. - PubMed

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