Quantifying the cluster of differentiation 4 receptor density on human T lymphocytes using multiple reaction monitoring mass spectrometry.

Cluster of differentiation 4 (CD4) is an important glycoprotein containing four extracellular domains, a transmembrane portion and a short intracellular tail. It locates on the surface of various types of immune cells and performs a 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 the 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, flow cytometry, the conventional method of quantification of the CD4 density on the T cell surface depends on antibodies and has suffered from variables such as antibody clones, the fluorophore and conjugation chemistries, the fixation conditions, and the flow cytometric quantification methods used. In this study, we report the development of a highly reproducible nano liquid chromatography-multiple reaction monitoring mass spectrometry-based quantitative method to quantify the CD4 receptor density in units 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.