Self-packed size-exclusion columns enable versatile high-throughput native, top-down, and ion mobility-mass spectrometry studies on proteins and complexes.

Native MS (nMS) is a key structural biology technique that makes it possible to study intact proteins and their interactions. Unfortunately, non-volatile salts are incompatible with nMS, which demands a laborious desalting procedure. Non-denaturing size-exclusion chromatography (SEC) allows both rapid desalting and separation and has previously been explored for nMS automation.

Mass Spectrometry Analysis of Chemically and Collisionally Dissociated Molecular Glue- and PROTAC-Mediated Protein Complexes Informs on Disassembly Pathways.

Molecular glues (MGs) and proteolysis-targeting chimeras (PROTACs) are used to modulate protein-protein interactions (PPIs), via induced proximity between compounds that have little or no affinity for each other naturally. They promote either reversible inhibition or selective degradation of a target protein, including ones deemed undruggable by traditional therapeutics. Though native MS (nMS) is capable of analyzing multiprotein complexes, the behavior of these artificially induced compounds in the gas phase is still not fully understood, and the number of publications over the past few years is still rather limited.

Cross-Interactions of Aβ Peptides Implicated in Alzheimer's Disease Shape Amyloid Oligomer Structures and Aggregation.

A defining hallmark of Alzheimer's disease (AD) is the synaptic aggregation of the amyloid β (Aβ) peptide. , Aβ production results in a diverse mixture of variants, of which Aβ40, Aβ42, and Aβ43 are profusely present in the AD brain, and their relative abundance is recognized to play a role in disease onset and progression. Nonetheless, the occurrence of Aβ40, Aβ42, and Aβ43 hetero-oligomerization and the subsequent effects on Aβ aggregation remain elusive and were investigated here.

Mass spectrometry reflects key aspects of copper-amyloid β chemistry.

Mass spectrometry is a powerful method to study protein complexes; however, biochemical reactions are typically beyond the scope of MS studies. Here, we have studied the gas-phase redox chemistry of the [copper(II) - amyloid β] complex and show that the sequence-dependence of this chemistry reflects key aspects of the known behaviour of different variants of the peptide.

Top-down mass spectrometry of native proteoforms and their complexes: a community study.

The combination of native electrospray ionization with top-down fragmentation in mass spectrometry (MS) allows simultaneous determination of the stoichiometry of noncovalent complexes and identification of their component proteoforms and cofactors. Although this approach is powerful, both native MS and top-down MS are not yet well standardized, and only a limited number of laboratories regularly carry out this type of research. To address this challenge, the Consortium for Top-Down Proteomics initiated a study to develop and test protocols for native MS combined with top-down fragmentation of proteins and protein complexes across 11 instruments in nine laboratories.

Top-down mass spectrometry of native proteoforms and their complexes: A community study.

The combination of native electrospray ionisation with top-down fragmentation in mass spectrometry allows simultaneous determination of the stoichiometry of noncovalent complexes and identification of their component proteoforms and co-factors. While this approach is powerful, both native mass spectrometry and top-down mass spectrometry are not yet well standardised, and only a limited number of laboratories regularly carry out this type of research. To address this challenge, the Consortium for Top-Down Proteomics (CTDP) initiated a study to develop and test protocols for native mass spectrometry combined with top-down fragmentation of proteins and protein complexes across eleven instruments in nine laboratories.

Covalent -Adenosylhomocysteine-Based DNA Methyltransferase 2 Inhibitors with a New Type of Aryl Warhead.

The DNA methyltransferase 2 (DNMT2) is an RNA modifying enzyme associated with pathophysiological processes, such as mental and metabolic disorders or cancer. Although the development of methyltransferase inhibitors remains challenging, DNMT2 is not only a promising target for drug discovery, but also for the development of activity-based probes. Here, we present covalent SAH-based DNMT2 inhibitors decorated with a new type of aryl warhead.

New Insights into the Intrinsic Electron-Based Dissociation Behavior of Cytochrome c Oligomers.

Between 2003 and 2017, four reports were published that demonstrated the intrinsic ability of the native iron-containing proteins cytochrome c and ferritin to undergo radical-based backbone fragmentation in the gas phase without the introduction of exogenous electrons. For cytochrome c in particular, this effect has so far only been reported to occur in the ion source, preventing the in-depth study of reactions occurring after gas-phase isolation of specific precursors. Here, we report the first observation of this intrinsic native electron capture dissociation behavior after quadrupole isolation of specific charge states of the cytochrome c dimer and trimer, providing direct experimental support for key aspects of the mechanism proposed 20 years ago.

Charge site manipulation to enhance top-down fragmentation efficiency.

In recent years, top-down mass spectrometry has become a widely used approach to study proteoforms; however, improving sequence coverage remains an important goal. Here, two different proteins, α-synuclein and bovine carbonic anhydrase, were subjected to top-down collision-induced dissociation (CID) after electrospray ionisation. Two high-boiling solvents, DMSO and propylene carbonate, were added to the protein solution in low concentration (2%) and the effects on the top-down fragmentation patterns of the proteins were systematically investigated.

Seeing the complete picture: proteins in top-down mass spectrometry.

Top-down protein mass spectrometry can provide unique insights into protein sequence and structure, including precise proteoform identification and study of protein-ligand and protein-protein interactions. In contrast with the commonly applied bottom-up approach, top-down approaches do not include digestion of the protein of interest into small peptides, but instead rely on the ionization and subsequent fragmentation of intact proteins. As such, it is fundamentally the only way to fully characterize the composition of a proteoform.

Bone Sialoprotein Immobilized in Collagen Type I Enhances Bone Regeneration In vitro and In vivo.

The use of bioactive molecules is a promising approach to enhance the bone healing properties of biomaterials. The aim of this study was to define the role of bone sialoprotein (BSP) immobilized in collagen type I in various settings. In vitro studies with human primary osteoblasts in mono- or in co-culture with endothelial cells demonstrated a slightly increased gene expression of osteogenic markers as well as an increased proliferation rate in osteoblasts after application of BSP immobilized in collagen type I.