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  • Glycosylation is arguably one of


    Glycosylation is arguably one of the most difficult PTMs to analyze via mass spectrometry. Unlike with acetylation or phosphorylation, where the addition of a standard group can be accounted for due to expected mass shifts, glycosylation occurs through the addition of varying isobaric glycans [35]. Because the glycans are so large (typically several thousand mass units), often times, peptide masses are shifted by such a large number, that sequencing is no longer viable. Therefore, it is often required to first remove the glycans and subsequently digest the protein for analysis [35]. The documented methods of proteomic analysis of glycoproteins is highly dependent on the type of glycosylation present. For instance, N-glycoproteins are typically enriched through hydrazide chemistry [41]. This enrichment has been followed by digestion, derivatization and mass spectrometric analysis to shed light on N-glycoprotein characterization and quantification in human saliva [42]. These methods have been shown to elicit insight into the glycosylation sites on the protein, however, because of the isobaric nature of the glycans, elucidation of structure yields further difficulties. Because AβPP has been documented to undergo N-glycosylation, methods such as these may begin to, not only confirm or deny the glycosylation sites proposed in earlier works [40], but quantify the glycan occupancy for the individual sites and relate that to disease pathology. A secondary approach to glycan capture, comes with the application of lectin nanoprobes [43]. This technique has been coupled with LC-MS/MS analysis to qualitatively and quantitatively characterization glycan modifications in complex human samples [44] which would be of benefit to the analysis of samples such as isolated NFTs and senile plaques.
    Amyloid-beta As previously mentioned, amyloid-beta peptides ranging from 40 to 42 amino acids in length are thought to be the primary component of senile plaque 2-NBDG structure in the brains of those with AD [7,45]. These extracellular protein aggregates are insoluble and although they have been thoroughly characterized by immunohistochemistry and fluorescent microscopy [7,46], there is still much left unknown about their mechanisms of aggregation and even their composition. Ubiquitin, an 8 kDa protein that is expressed in most eukaryotic cells [28], has previously been detected within neurofibrillary tangles (NFTs) and senile plaques in AD brains [29]. Ubiquitination induces protein degradation, disrupts protein-protein interactions, and modifies the localization of proteins [47,48]. It occurs through the covalent attachment of one or more ubiquitin molecules to a target protein [1]. This modification occurs in three steps. First, a high-energy thiol ester intermediate is formed by an activated C-terminal glycine of a ubiquitin molecule. Next, the activated ubiquitin is transferred to a member of the ubiquitin-protein ligase family which then promotes the transfer of the ubiquitin molecule to a lysine residue of the target protein [1]. Defects in this pathway have been documented to be associated with the etiology of various neurological disorders [49]. Because the pathway is important in the removal of misfolded proteins and many neurological disorders are characterized by the build-up of misfolded proteins, it is logical that disruptions in the ubiquitination pathway could decrease the ability to transport and degrade misfolded aggregates. Because of the focus on amyloid-beta in AD research, PTM analysis by mass spectrometry as it pertains to AD has been accomplished and success has been found in better understanding the roles of modifications in amyloid-beta peptides. Tay et al. (2012) characterized the PTMs associated with amyloid-beta by mass spectrometry [50]. Mass spectrometry methodology called precursor ion mapping (PIM) was described as a means of modification characterization. The synthetic amyloid-beta [[1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40]] monomer was cross-linked and subsequently digested, yielding three transglutaminase cross-linked species [50]. The undigested protein did not yield these fragments. This technique had previously been described as a means of phosphorylation, oxidation, and glycosylation analysis [[51], [52], [53]]. The use of the synthetic peptide as a model compound suggests that this method would also be of benefit for the analysis of endogenous human peptides and proteins. Inoue et al. (2013) described a covalent chiral derivatized ultraperformance liquid chromatography tandem mass spectrometry (CCD-UPLC-MS/MS) method for determining amino acid racemization (AAR) and amino acid isomerization (AAI) of N-terminal amyloid-beta [[1], [2], [3], [4], [5]] in human brain. Quantification was accomplished with a stable isotope (15 N) labeled amyloid-beta [[1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40]] as an internal standard [54]. We have developed mass spectrometry-based methods for the analysis of amyloid-beta [55,56] and modified amyloid-beta [57]. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) methods that we have developed have the potential to be applied to the characterization of other forms of modified amyloid-beta.