My research focuses on quantification and characterization of membrane-associated proteins (MAPs) in complex biological samples using liquid chromatography (LC) coupled tandem mass spectrometry (MS) with isotope labeled internal standards. By using our optimized sample preparation protocol combined with nanoLC - multiple reaction monitoring (MRM) /MS, we successfully performed quantitative analysis for multiple target MAPs, including the Alzheimer's disease risk factor apolipoprotein E4 (apoE4) isoform from human frontal cortex, the clinical cell surface marker cluster of differentiation 4 (CD4) from human T lymphocytes and cholesterol-metabolizing cytochrome P450s with their redox partners from human temporal lobe and retina. Our study addressed the following issues: 1) development of various types of isotope-labeled internal standards for quantification of the target MAPs; 2) quantitative differentiation of specific protein isoforms; and 3) quantitative analysis of protein cleavage and fragment accumulation associated with human disease. We demonstrate that our quantitative workflow based on LC-MRM/MS can be used for absolute quantification of MAPs as well as for the exploration of basic mechanisms of human diseases. We believe this method can be widely applied for other MAPs and MAP-related protein analyses.
Biomolecular Measurement Division
Bioanalytical Science Group