(6) Abnormally phosphorylated and otherwise post-translationally modified tau has also received significant attention, as it is a primary constituent of neurofibrillary tangles (NFTs) and neuropil threads that are observed within neurons in partially overlapping brain regions of people with AD. Many hypotheses have focused on aggregation of Aβ peptides, which form toxic oligomers as well as larger fibrils and ultimately extracellular deposits in multiple brain regions of people with AD. Despite decades of intense investigation, the underlying causes of AD remain incompletely understood. If the extent of amino acid isomerization in biological samples could be easily determined by DIA, then connections to the origin of AD and other age-related diseases could be investigated more easily. (4) Some progress has been made with identification of phosphorylation isomers, though these isomers can be distinguished by many unique mass fragments. In theory, DIA data should contain information about isomers, but this capability has not been demonstrated because typical DIA analysis pipelines only identify the best peak for any given sequence. Identification of isomers in DDA is further complicated by dynamic exclusion, which precludes examination of the same mass for a defined period of time. An example of isomerization at Asp for the tryptic peptide 386TDHGAEIVYK 395 from tau is illustrated in Scheme 1. (3) Mass spectrometric analysis of peptide isomers (where typically L-Asp has been swapped for D/L-isoAsp) and epimers (where a single residue has been flipped from the L to D configuration) is typically stymied by several complicating factors including no mass change following modification, absence of unique or characteristic fragments, and similarity of fragmentation patterns in general. (1,2) Recent work has posited a connection between Alzheimer’s disease (AD) and isomerization of amino acids in long-lived proteins, which may interfere with lysosomal digestion. Despite the potential for reanalysis of DIA data, examples are limited. This advantage is anticipated because DIA experiments are designed to collect information about all species eluting from chromatographic separation without selection as occurs in data-dependent acquisition (DDA). In this case, a promising target for future investigations into the therapy and prevention of AD has been identified.Ī frequently mentioned advantage of data-independent acquisition (DIA) proteomics approaches is the potential capture of information not originally sought when then data were acquired. This study illustrates that DIA data may contain unforeseen results of interest and may be particularly useful for pilot studies investigating new research directions. These results suggest, but do not conclusively demonstrate, that lower autophagic flux may be strongly associated with loss of function in AD brains. Differences consistent with reduced autophagic flux in AD-related samples relative to controls were found for numerous proteins, including most notably p62, a recognized indicator of autophagic inhibition. To explore potential mechanisms that might account for these observations, quantitative analysis of proteins related to isomerization repair and autophagy was performed. Relative to controls, a surprising increase in isomer abundance was found in both autosomal dominant and sporadic AD samples. Examination of a large set of human brain samples revealed a striking relationship between Alzheimer’s disease (AD) status and isomerization of aspartic acid in a peptide from tau. Herein, we reanalyzed DIA data originally recorded for global proteomic analysis to look for isomerized peptides, which occur as a result of spontaneous chemical modifications to long-lived proteins. One of the potential benefits of using data-independent acquisition (DIA) proteomics protocols is that information not originally targeted by the study may be present and discovered by subsequent analysis.
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