Method for quantification of low copy number proteins using single molecule spectroscopy applied to synaptic vesicle membrane proteins

This dissertation presents a technique which utilizes single molecule fluorescence to quantify synaptic vesicle membrane proteins. The average number and distribution in the number of seven synaptic vesicle integral proteins was determined by deconvolving intensity distributions of labeled synaptic vesicles. The theory of deconvolution and the method of analysis is presented. The vesicles were quantitatively labeled using primary antibodies directed at the vesicle protein being measured. The number of several vesicle proteins reported, including SV2, the proton ATPase, V-glut1, and synaptotagmin, show little variation between vesicles, indicating that they are sorted to vesicles with high precision. The number of other proteins, including VAMP, synaptophysin and synaptogyrin, is distributed more broadly, consistent with stochastic sorting. These findings suggest that variation in the number of tightly sorted proteins could lead to pathological changes in synaptic functioning, whereas variability in the number of more randomly-sorted proteins might encode normal changes in vesicle fusion that contribute to synaptic plasticity. This is the first comprehensive single-molecule quantification of integral membrane proteins in an isolated organelle. This method is expected to be of broad use in quantitative cell biology.