The precise mechanisms through which post-translational modifications modulate protein function are not clear nor is it understood how they are implicated in human diseases at the molecular level. The objective of this proposal is to develop a method that reveals how variations in sequence and post-translational modifications modulate the structural heterogeneity of proteins. Guided by our strong preliminary data, we will obtain the objective of this proposal by pursuing the following specific aims: 1) To enable structure-elucidation of differentially modified proteins by ion mobility spectrometry / mass spectrometry; and 2) To characterize the structural heterogeneity of differentially modified proteins by tandem-trapped ion mobility spectrometry / mass spectrometry. In the first Aim, we will develop a method that determines structures for differentially modified proteins and their assemblies, in particular for phosphorylated and glycosylated species. In the second Aim, we will develop an approach that reveals how protein structure depends on amino acid sequence and post- translational modifications. The research proposed in this application is innovative because it substantially advances from the status quo through unique computational and experimental methods that were recently developed in our lab, namely the Structure Relaxation Approximation and tandem-trapped ion mobility spectrometry/mass spectrometry methods. This contribution is significant because it is the first step towards a general analytical method that is expected to provide a molecular-level understanding of how changes in amino acid sequence and post-translational modifications are implicated in disease mechanisms. Ultimately, the results of the proposed work can be expected to significantly benefit a number of research areas relevant to the mission of the NIH, including the development of a vaccine against HIV/AIDS.