Date of Degree
Emmanuel J. Chang
Mass spectrometry, phosphorylation, quantification
Protein phosphorylation modification regulates numerous cellular functions by a reversible and selective control of kinases and phosphatases. To understand the entire dynamic network of phosphorylation requires sensitive and reliable quantification of phosphorylation, measurements that can be achieved by mass spectrometry. In this research, we established efficient MALDI-mass spectrometric methods as strategies for single- or multi-site phosphorylation quantification without the use of isotopes, chromatography and calibration curves. The methods were assessed by analyzing peptide standards with different single-multiple phosphorylation sites, showing a wide dynamic range, good accuracy and reproducibility. This is the first label-free MALDI method without using a calibration methodology proposed for quantification of in vitro phosphorylation in a kinase assay.
Moreover, advanced mass spectrometry empowers identification of a highly conserved Cdk2 phosphorylation site of HIV-1 reverse transcriptase (RT) at Thr 261 across thousands of HIV-1 strains. We demonstrated phosphorylation on HIV-1 RT peptides and protein in in vitro assays, and confirmed phosphorylation in vivo with antibodies and mutation studies. Blocking this phosphorylation by p21, a naturally occurring Cdk inhibitor, defines a potential Cdk2-mediated cell-intrinsic mechanism for restricting HIV-replication in a clinically significant way.
Ho, Hsin-Pin, "Development and Applications of Mass Spectrometric Methods for Phosphorylation Analysis" (2014). CUNY Academic Works.