The linear sequence of amino acids in a protein folds into a 3D structure to execute protein activity and function, but it is still challenging to profile the 3D structure at the proteome scale. Here, we present a method of native protein tandem mass tag (TMT) profiling of Lys accessibility and its application to investigate structural alterations in human brain specimens of Alzheimer's disease (AD). In this method, proteins are extracted under a native condition, labeled by TMT reagents, followed by trypsin digestion and peptide analysis using two-dimensional liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS). The method quantifies Lys labeling efficiency to evaluate its accessibility on the protein surface, which may be affected by protein conformations, protein modifications, and/or other molecular interactions. We systematically optimized the amount of TMT reagents, reaction time, and temperature and then analyzed protein samples under multiple conditions, including different labeling time (5 and 30 min), heat treatment, AD and normal human cases. The experiment profiled 15370 TMT-labeled peptides in 4475 proteins. As expected, the heat treatment led to extensive changes in protein conformations, with 17% of the detected proteome displaying differential labeling. Compared to the normal controls, AD brain showed different Lys accessibility of tau and RNA splicing complexes, which are the hallmarks of AD pathology, as well as proteins involved in transcription, mitochondrial, and synaptic functions. To eliminate the possibility that the observed differential Lys labeling was caused by protein level change, the whole proteome was quantified with standard TMT-LC/LC-MS/MS for normalization. Thus, this native protein TMT method enables the proteome-wide measurement of Lys accessibility, representing a straightforward strategy to explore protein structure in any biological system.