Developing mass spectrometry application in quantification and characterization of cell membrane associated proteins in human disease

 

Meiyao Wang1, 2 and Illarion V. Turko1, 2

1Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850
2 Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD 20899

We report using mass spectrometry based method as a major tool for: 1) quantification of membrane associated proteins; 2) differentiation of specific isoform protein based on unique amino acid sequence; and 3) characterization of structure associated protein behavior in Alzheimerís disease (AD).

 

Our study took the major AD risk factor protein, apolipoprotein E (Apo) isoform E4, as the target molecule to develop a strategy of using quantitative mass spectrometry to characterize proteinís roles associated with human disease. The major isoforms of ApoE protein include ApoE2, ApoE3 and ApoE4, which are only different by arginyl/cysteinyl at two positions on protein sequence. However, these minor distinctions in sequence lead to different protein functions in neurobiology. It is difficult or even impossible to differentiate or quantify the isoforms from each other by conventional antibody-based method. We successfully achieved that goal by applying nanoflow liquid chromatography (nanoLC) coupled high specific multiple reaction monitoring mass spectrometry (MRM MS), supplied with isotope labeled full-length protein standard.

 

Our results of protein quantitative measurements indicated AD associated protein fragmentation and accumulation of ApoE protein. It demonstrates that quantitative MRM mass spectrometry can be useful for better understanding protein structure associated basic mechanism of human disease. This method can be widely applied for other membrane protein studies.