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  • While mass spectrometry is now routinely utilized


    While mass spectrometry is now routinely utilized to investigate PTMs, it is more often times used for the development of antibodies and antibody-enrichment strategies for the observation of PTMs with low site occupancy [36]. This is particularly important when the sample medium becomes complex, such as with tissue homogenates, blood, or cerebrospinal fluid (CSF). These enrichment strategies work well when it comes to modification specific analysis (for instance if all phosphorylation sites are of interest etc.). However, other approaches must be utilized when the target analyte is a specific protein or all potential modifications, even those of low occupancy, are to be analyzed. Database searches are routinely utilized to identify proteins by their peptide fingerprint after MS/MS analysis. Enzymatically digested peptides and their fragments are analyzed for their amino 2-NBDG chemicals sequence and the probability of protein identification is given at a user specified error [35]. Additionally, the probability of modifications can be specified within the search criteria to retrieve identifications containing the expected modification [35]. Typically, however, the modifications must be amino acid-specific and singular. Multiple modification sites on a single peptide, or a non-specific modification can be difficult to identify by database searches due to their reliance on probability and theoretical enzymatic peptides of the protein hits [35]. A work-around for this, is the manual analysis of MS/MS spectra with special attention to the peptide fragment mass shifts commonly reported for the modification. The intact parent mass (m/z) of the peptide will be shifted by a specific number of Da (as will all modification-containing fragments). One must therefore assume, that the modification containing peptide is stable enough to withstand the conditions of MS/MS analysis [35]. This becomes tricky when modifications such as glycosylation, which will be discussed in more detail later, are of interest. Table 1 lists some relevant PTMs and the corresponding average mass shifts commonly observed in the mass spectra.
    Amyloidβ protein precursor (AβPP) The AβPP is an integral type 1 membrane protein, and while its exact function is debated, in the healthy state, it has roles in synapse formation and neuronal plasticity [37]. It is because of this that mutations of normal AβPP cleavage have been linked to AD. AβPP is cleaved in three different ways, by α-, β-, and/or γ-secretase. Intracellular cleavage occurs through α-secretase. AβPP is cleaved at the -carboxyl end of the protein near the cellular membrane by the α-secretase enzyme that results in the creation of a larger intracellular protein which is thought to be neuroprotective. AβPP is also cut by β-secretase and γ-secretase in the absence of α-cleavage to yield the small 40–42 amino acid sized Aβ peptide which accumulates into aggregates and plaques [[37], [38], [39]]. This section will focus on the modifications linked to AβPP and amyloid-beta peptides and the PTMs associated with them will be discussed in more detail in the following section. Many AD studies have focused on the suspected neurotoxic amyloid-beta [[40], [41], [42]] species. It is therefore important to first better understand the mechanisms of AβPP modification. If variations surrounding AβPP processing are clarified, it is possible that preventative therapies could be developed. Several PTMs have been observed to occur during the translation of AβPP [1,5,7,40]. Some of these modifications contribute to normal AβPP pathology. However, a change in the homeostasis of the modifications can have either negative or positive effects that may then contribute to, or theoretically protect from, developing AD [40]. AβPP has two possible N-glycosylation sites [40]. It has been documented that an imbalance in the proper glycosylation form of the protein can inhibit the secretion of the neuroprotective form of the protein and facilitate the build-up of the cellular protein [40]. On the other hand, if an additional terminal sialic acid is added to the oligosaccharide chain of the protein, it has been reported that the secretion of the soluble, neuroprotective protein is increased [40].