Detection of Amino Acid Isomers via Data-Independent Acquisition Enables Observation of Proteome Aging in Human Lens Tissue

In proteins, individual amino acids may spontaneously isomerize, particularly aspartic acid. This inevitable modification can inhibit proteolysis and even negatively affect protein function. Unfortunately, amino acid isomerization is difficult to detect via mass spectrometry. Without any shift in mass or sequence, there is no change to m/z for either precursors or b/y fragments. Despite this, LC-MS utilizing data-independent acquisition (DIA) has emerged as an effective way to detect large numbers of isomerization sites across entire proteomes. The structural changes following spontaneous isomerization are generally enough to significantly shift peptide retention time. In DIA data, this translates to chromatograms containing multiple peaks, each representing distinct isomer subpopulations. These separated peaks can then be integrated to quantify the extent of isomerization. Together, this allows for easy detection and analysis of isomers. The human lens proteome is not subject to normal proteostasis, and most proteins in the lens have lifetimes on par with the age of their owner. Because of this, slow and spontaneous changes like isomerization are capable of accumulating to a greater degree in the lens than in other proteomes. However, the study of isomerization in the lens has been nearly entirely relegated to crystallins, a class of proteins that make up >90% of all lens protein by mass. Using DIA and non-crystallin protein enrichment, we have done a far more extensive search for isomer sites in a large cohort of lens samples. This search has yielded hundreds of aspartic acid isomer sites in the inner nucleus, outer nucleus, and cortex of 15 subjects ranging from 15 to 74 years old. Quantification of this isomerization clearly shows the effects of aging on isomerization, as well as differences in isomerization across each lens region.