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  • In addition to phosphorylation tau has also been shown


    In addition to phosphorylation, tau has also been shown to undergo acetylation [14,15,74]. Acetylation occurs when the acetyl-coenzyme A transfers an acetyl group to the ε-amino group of a lysine residue of a target protein [73]. This PTM is reversible and catalyzed by histone acetyl transferases (HATs) and histone de-acetylases (HDACs). Like phosphorylation, an imbalance in the two Filipin Complex (HAT/HDAC) equilibrium can cause a disruption in homeostasis of the central nervous system [73]. Acetylated tau has been detected to exclusively occur in the brains of those with various tauoapthies [73]. Lys174, for instance, was characterized as an acetylation site during early stages of AD using ESI MS/MS of cell lysates from AD brains [14]. Both phosphorylation and acetylation can reduce tau affinity for microtubules and thus facilitates tau aggregation [14]. Additionally, it has been documented that tau acetylation can cause hyperphosphorylated tau aggregation due to lowered protein degradation [74]. Acetylation most commonly occurs on the lysine and arginine amino groups and exhibits a characteristic 42 Da mass shift [33]. While phosphorylation is one of the most common modifications linked to AD, it can be one of the most difficult to analyze by mass spectrometry. Phosphopeptides are typically present in low quantities compared to other peptides in a biological sample and are therefore particularly susceptible to ion suppression [35]. It has been reported that the more phospho-groups a peptide has, the lower the sensitivity for this PTM [75,76]. Although, this phenomenon has been challenged. In a recent work, the correlations of the extent of phosphorylation and signal intensity were investigated by MALDI and ESI. Mass spectra of three peptide standards containing zero to three phosphotyrosines were collected and analyzed and the spectra originating from ESI and MALDI each showed a decrease in signal intensity with increasing phosphorylation [35]. Due to the labile nature of phosphoryl groups, it is not uncommon for modified peptides to lose sites of phosphorylation during the ionization process. According to Parker et al., this can partially be accounted for when using MALDI as the mode of analysis. Some organic matrices (such as 2,5- dihydroxybenzoic acid – DHB) transfer less energy to the analyte and are therefore more suitable for phosphopeptide analysis [35]. Phosphorylation is typically identified by a characteristic 80 Da shift in the mass of the modified peptide although it is common practice to first attempt to enrich the sample for the phosphopeptides of interest due to their low abundance. However, there is no standard protocol for doing this, as different phosphopeptides require different steps for enrichment. A secondary technique is to compare spectra of peptides before and after being treated with phosphatase which will result in a loss of 80 Da from those signals corresponding to phosphorylated peptides [34,77]. Quantification of PTMs may be one of the most important aspects of analyzing biological samples. As previously mentioned, protein modifications are not all negative and their regulation is important in normal biological function. Multiple reaction monitoring (MRM) is the main MS-based technology utilized to simultaneously quantify multiple modified peptides in a sample [33,34,36,73]. This, and similar mass spectrometric techniques, have been previously utilized in the determination of the abundance of the polyubiquination of tau in human AD [66] and the abundance of tau and amyloid-beta in human CSF [[78], [79], [80], [81]]. Thomas and Yang [73] outline a thorough means of identifying and quantifying lysine-residue modifications in tau. Their method however, does not specify an exact means of tau isolation. Although, it is strongly suggested that, at a minimum, 20 μg of purified tau is used as starting material. It is well documented that the isolation and purification of tangles from AD brains is non-trivial [[82], [83], [84]]. Additionally, great care should be taken during isolation procedure because solvents utilized for preparation and storage can have negative effects on the mass spectra produced [57,85,86].