QuAntitative Measurement of CD4 protein on Human T cell surface using Multiple Reaction Monitoring Mass Spectrometry

Meiyao Wang1, 2, Hua-Jun He3, Illarion V. Turko1, 2 and Lili Wang3

1Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850
2Analytical Chemistry Division, 3Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, MD 20899

 

Since CD4 protein expression on human T cell surface has been used as a biological calibrator for quantification of other cell surface antigens using flow cytometry, multiple antibody-based methods have been developed and used for quantification of CD4 protein. However, the CD4 protein density values reported in literature vary significantly due to variables such as antibody clones, conjugations chemistries and fixation conditions.   

In this study, we have developed an absolute quantification method for CD4 receptor on T cell surface using Multiple Reaction Monitoring Mass Spectrometry (MRM-MS) combined with the 15N13C-isotope labeled recombinant CD4 protein as an internal standard. This MRM-MS based approach enables the quantitative measurement of cell surface protein without the use of an antibody. Multiple CD4 signature peptides were employed to quantify the protein expression levels in cell lysates with known numbers of CD4+ cells. Statistical analysis was performed based on multiple biological replications and technical repeats. The results from the MRM-MS method and the conventional flow cytometry method were also compared.

The MRM-MS based method, as an effective validation method, provides a complementary mean to the antibody-based flow cytometric method for quantifying the cell surface and intracellular proteins. The study verifies MRM-MS as a potential clinical laboratory tool for the measurements of cell protein markers.