Neurodegenerative Proteinopathies in the Proteoform Spectrum-Tools and Challenges

Abstract

Proteinopathy refers to a group of disorders defined by depositions of amyloids within living tissue. Neurodegenerative proteinopathies, including Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, and others, constitute a large fraction of these disorders. Amyloids are highly insoluble, ordered, stable, beta-sheet rich proteins. The emerging theory about the pathophysiology of neurodegenerative proteinopathies suggests that the primary amyloid-forming proteins, also known as the prion-like proteins, may exist as multiple proteoforms that contribute differentially towards the disease prognosis. It is therefore necessary to resolve these disorders on the level of proteoforms rather than the proteome. The transient and hydrophobic nature of amyloid-forming proteins and the minor post-translational alterations that lead to the formation of proteoforms require the use of highly sensitive and specialized techniques. Several conventional techniques, like gel electrophoresis and conventional mass spectrometry, have been modified to accommodate the proteoform theory and prion-like proteins. Several new ones, like imaging mass spectrometry, have also emerged. This review aims to discuss the proteoform theory of neurodegenerative disorders along with the utility of these proteomic techniques for the study of highly insoluble proteins and their associated proteoforms.

Keywords: 2D-PAGE; hydrogen/deuterium exchange mass spectrometry; imaging MS; prion-like proteins; proteinopathies; proteoforms; top-down MS.

Figure 1

The mechanism of formation of amyloids: The organization of proteins into globular, partially folded or intrinsically disordered functional conformations is a tightly regulated process. However, mutations, aberrant cleavage or cellular stress can cause the formation of altered monomeric species. This event, known as monomer activation, destabilizes the native structure and forms the thermodynamically favorable β-sheets rich structure. The combination of these altered structures, or primary nucleation, leads to the formation of various multimers, protofibrils (2.5 to 3 nm in diameter), and fibrils (6 to 10 nm). The intertwining of protofibrils and fibrils leads to the formation of highly stable mature amyloid fibrils (60–120 nm). The primary event of nucleation and fibril formation is relatively slow and is referred to as the lag phase of growth. As more native proteins mimic the structure of misfolded seeds, the growth of amyloid fibrils reaches an exponential phase leading to rapid accumulation of aggregates [7,8].

Figure 2

Involvement of known prion-like proteins in multiple neurodegenerative disorders. The figure depicts the overlapping pathological profile of PrP (green circle), α-Synuclein (red circle), Aβ (blue circle), Tau (yellow circle), and TDP-43 (black circle). Each of the stated disorders have further clinical variants (as shown in the case of AD), thereby complicating the role of prion-like proteins in bringing about the observed pathology. PDD—Parkinson’s disease with dementia; DS—Down’s syndrome; FTD-T—frontotemporal dementia with tau pathology; fAD—familial AD; sAD—sporadic AD; rpAD—rapidly-progressive AD; PCA-AD—posterior cortical atrophy–AD; PPA-AD—primary progressive aphasia with AD.

Figure 3

Virtual 2D map for AD-associated prion-like proteins. Differences in electrophoretic mobility of Aβ proteoforms based on variations in post-translational cleavage.

Figure 4

Summarized methodology matrix-assisted laser desorption ionization-imaging mass spectrometry (MALDI-IMS) for Aβ proteoforms. A section of the tissue is deposited onto conductive slides (step 1) and coated uniformly with the appropriate matrix solution (step 2). Matrix and sample are desorbed and ionized upon the application of ultraviolet laser (red line; step 3). These gaseous ions (colored dots) are then analyzed. Targeted proteins can be localized by selecting the m/z value corresponding to their ions as shown for the m/z ratio of 786, 3333.3, 4556.3 and 1553.3 with green stars in the figure. The relative number of stars depicts the hypothetical signal intensity for the selected m/z ratio.