Protein glycosylation is a highly important, yet poorly understood protein post-translational modification. Thousands of possible glycan structures and compositions create potential for tremendous site heterogeneity. A lack of suitable analytical methods for large-scale analyses of intact glycopeptides has limited our abilities both to address the degree of heterogeneity across the glycoproteome and to understand how this contributes biologically to complex systems. Here we show that N-glycoproteome site-specific microheterogeneity can be captured via large-scale glycopeptide profiling methods enabled by activated ion electron transfer dissociation (AI-ETD), ultimately characterizing 1,545 N-glycosites (>5,600 unique N-glycopeptides) from mouse brain tissue. Our data reveal that N-glycosylation profiles can differ between subcellular regions and structural domains and that N-glycosite heterogeneity manifests in several different forms, including dramatic differences in glycosites on the same protein. Moreover, we use this large-scale glycoproteomic dataset to develop several visualizations that will prove useful for analyzing intact glycopeptides in future studies.
Myocardial fuel and energy metabolic derangements contribute to the pathogenesis of heart failure. Recent evidence implicates posttranslational mechanisms in the energy metabolic disturbances that contribute to the pathogenesis of heart failure. We hypothesized that accumulation of metabolite intermediates of fuel oxidation pathways drives posttranslational modifications of mitochondrial proteins during the development of heart failure. Myocardial acetylproteomics demonstrated extensive mitochondrial protein lysine hyperacetylation in the early stages of heart failure in well-defined mouse models and the in end-stage failing human heart. To determine the functional impact of increased mitochondrial protein acetylation, we focused on succinate dehydrogenase A (SDHA), a critical component of both the tricarboxylic acid (TCA) cycle and respiratory complex II. An acetyl-mimetic mutation targeting an SDHA lysine residue shown to be hyperacetylated in the failing human heart reduced catalytic function and reduced complex II–driven respiration. These results identify alterations in mitochondrial acetyl-CoA homeostasis as a potential driver of the development of energy metabolic derangements that contribute to heart failure.
Site-specific
characterization of glycosylation requires intact
glycopeptide analysis, and recent efforts have focused on how to best
interrogate glycopeptides using tandem mass spectrometry (MS/MS).
Beam-type collisional activation, i.e., higher-energy collisional
dissociation (HCD), has been a valuable approach, but stepped collision
energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental
activation (EThcD) have emerged as potentially more suitable alternatives.
Both sceHCD and EThcD have been used with success in large-scale glycoproteomic
experiments, but they each incur some degree of compromise. Most progress
has occurred in the area of N-glycoproteomics. There
is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance
for the two classes of glycopeptides. Here, we systematically explore
the advantages and disadvantages of conventional HCD, sceHCD, ETD,
and EThcD for intact glycopeptide analysis and determine their suitability
for both N- and O-glycoproteomic
applications. For N-glycopeptides, HCD and sceHCD
generate similar numbers of identifications, although sceHCD generally
provides higher quality spectra. Both significantly outperform EThcD
methods in terms of identifications, indicating that ETD-based methods
are not required for routine N-glycoproteomics even
if they can generate higher quality spectra. Conversely, ETD-based
methods, especially EThcD, are indispensable for site-specific analyses
of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric
methods that are sufficient for N-glycopeptides,
and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.
Glycosylation is a prevalent, yet heterogeneous modification with a broad range of implications in molecular biology. This heterogeneity precludes enrichment strategies that can be universally beneficial for all glycan classes. Thus, choice of enrichment strategy has profound implications on experimental outcomes. Here we review common enrichment strategies used in modern mass spectrometry (MS)-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography (HILIC) and its derivatives, porous graphitic carbon (PGC), reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. Interest in glycoproteomics continues to surge as MS instrumentation and software improve, so this review aims to help equip researchers with necessary information to choose appropriate enrichment strategies that best complement these efforts.
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