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Quantifying CD4 Receptor Density on Human T Lymphocytes Using Multiple Reaction Monitoring Mass Spectrometry



Meiyao M. Wang, Hua-Jun He, Illarion Turko, Karen W. Phinney, Lili Wang


CD4 is an important glycoprotein containing a transmembrane portion and a short intracellular tail. It locates on the surface of various types of immune cells and performs critical role in multiple cellular functions such as signal amplification and activation of T cells. It is well known as a clinical cell surface protein marker for study of HIV progression and for defining T helper cell population in immunological applications. Moreover, CD4 protein has been used as a biological calibrator for quantification of other surface and intracellular proteins. However, the conventional method of quantification of CD4 density on T cell surface using flow cytometry depends on antibodies, which has suffered from the variables on antibody clones, fluorophore and conjugation chemistries, fixation conditions, and flow cytometric quantification methods used. In this study, we report the development of a highly reproducible nanoLC MRM MS-based quantitative method to quantify CD4 receptor density in the unit of copy number per cell on human CD4+ T cells. The method utilizes stable isotope labeled full-length standard CD4 as an internal standard to measure endogenous CD4 directly, without the use of antibodies. The development of the mass spectrometry-based approach of CD4 protein quantification is important as a complementary strategy to validate the analysis from the cytometry-based conventional method. It also provides new support for quantitative understanding and advanced characterization of CD4 on CD4+T cells.
Analytical Chemistry


CD4, T cells, multiple reaction monitoring mass spectrometry, absolute quantification, and isotope labeled standard


Wang, M. , He, H. , Turko, I. , Phinney, K. and Wang, L. (2013), Quantifying CD4 Receptor Density on Human T Lymphocytes Using Multiple Reaction Monitoring Mass Spectrometry, Analytical Chemistry, [online], (Accessed July 23, 2024)


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Created January 2, 2013, Updated October 12, 2021