Thomas et al aimed to utilize nanoflow LC
Thomas et al. (2012) aimed to utilize nanoflow LC-MS/MS to characterize modifications of tau . The resulting mass spectra identified a monomethylation of lysine which had never been reported. This methylation occurred across 7 amino acids, all of which were part of the microtubule-binding region and one of which was a competing site for ubiquitylation . To confirm the presence of these modified sites, post-mortem AD tissue was subject to confocal fluorescence microscopy with the addition of anti-tau and anti-methyl lysine antibodies . This paper is a prime example of the ways in which mass spectrometry was able to give more insight into the characterization of the PTM. The subsequent microscopy was critical to confirmation, however.
α-Tubulin While most researchers agree that hyperphosphorylated tau causes a breakdown of microtubules, evidence that this reduction correlates to an increase of PHFs is lacking. This suggests that other modification mechanisms are occurring in conjunction with the hyperphosphorylation of tau that lead to PHF build-up . Zhang et al. (2015) focused on the modifications associated with tubulin dimers to investigate how they affect the stability of the microtubules . It was confirmed from previous studies that the abundance of α-tubulin in the brains of those with AD is significantly decreased. All modified forms of α-tubulin (polyglutamated, tyrosinated, detyrosinated etc.) were also decreased. However, the proportion of acetylated α-tubulin to other forms of the protein was increased . This was determined by immunocytochemistry (using mouse antibodies for the various modifications being investigated), double-label immunofluorescence imaging (using inverted fluorescence and inverted laser-scanning confocal fluorescence microscopy), and western blotting. While the data is convincing, particularly due to the fact that it corroborates previously published results, advancements in mass spectrometry have been shown to be a robust means of characterizing and analyzing tubulin [, , ]. Additionally, it has been shown that SRM or MRM are more informative, primarily when it comes to quantification and highly complex sample analysis, than western blot analysis alone, although they may still be used in conjunction [90,91].
PTMs associated with oxidative stress Oxidative stress can compromise many biological molecules through redox reactions [92,93]. Additionally, various forms of oxidative stress can trigger PTMs by oxidation of amino Filipin Complex side chains [8,94]. Oxidative stress encompasses any imbalance in processes that cause the formation of harmful free radical species such as O2−, HO, and H2O2 . Although, in the normal state, the function and removal of these species is strictly regulated, in disease states such as with AD, oxidative stress can lead to the unintended modifications of proteins resulting in the misfolding of these proteins and eventual neuronal death [8,92,95,96]. The role of oxidative stress in neuronal disease and aging first became apparent in the early 1990s. A branch of the oxidative stress theory centralizes on the aggregation of amyloid-beta through dityrosine cross-links produced by Cu-mediated redox chemistry . There is significant prior work that suggests that Cu(II) binds to histidine and tyrosine amino acid residues on the amyloid-beta backbone [57,, , ]. In the presence of an oxidant (H2O2), it is thought that the Cu(II) mediates tyrosine cross-linking that potentially aids in the aggregation of amyloid-beta peptides and strengthens the insolubility of the structure , while yielding reactive oxygen species (ROS) polymerization that cause oxidative cell death . However, a recent mass spectrometry-based investigation into the dityrosine cross-links produced by reactive oxidation of amyloid-beta peptides was accomplished with the omission of the transition metal . Currently, there is equivocal evidence that the metal species (Cu, Zn etc.) play a significant role in the aggregation of amyloid-beta or other proteins in human brains. Furthermore, damage by free radicals has been implicated in AD pathology due to the elevated source from microglia [102,103], amyloid-beta [104,105], elevated levels of neuronal iron [, , ], and general imbalance in the homeostasis of metal ions . Silva et al. published a comprehensive table of common oxidative modifications and the common mass shifts associated to them . Like some of the other modifications discussed, oxidative modifications are low in abundance and unstable. Additionally, it is possible to observe oxidation of analytes caused by MS analysis . A positive aspect of MS analysis over other forms analysis, comes from the ability to account for modifications in database searches and use common mass shifts for identification with nonspecific modification analysis. We have already demonstrated the usefulness of MALDI-TOF MS for the analysis of metal-bound amyloid-beta  and are able to use the methods developed to analyze other oxidative modifications directly from complex human samples, such as isolated senile plaques, blood and CSF.