Electrospray ionization mass spectrometry (ESI-MS) is nowadays one of the cornerstones of biomolecular mass spectrometry and proteomics. Advances in sample preparation and mass analyzers have enabled researchers to extract much more information from biological samples than just the molecular weight. In particular, relevant for structural biology, noncovalent protein–protein and protein–ligand complexes can now also be analyzed by MS. For these types of analyses, assemblies need to be retained in their native quaternary state in the gas phase. This initial small niche of biomolecular mass spectrometry, nowadays often referred to as “native MS,” has come to maturation over the last two decades, with dozens of laboratories using it to study mostly protein assemblies, but also DNA and RNA-protein assemblies, with the goal to define structure–function relationships. In this perspective, we describe the origins of and (re)define the term native MS, portraying in detail what we meant by “native MS,” when the term was coined and also describing what it does (according to us) not entail. Additionally, we describe a few examples highlighting what native MS is, showing its successes to date while illustrating the wide scope this technology has in solving complex biological questions. Graphical Abstractᅟ
The Mla pathway is believed to be involved in maintaining the asymmetrical Gramnegative outer membrane via retrograde phospholipid transport. The pathway is composed of 3 components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here we solve the crystal structure of MlaC in its phospholipid free closed apo conformation, revealing a pivoting βsheet mechanism which functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC we provide evidence that the inner membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provides evidence of an apparatus for lipid export away from the inner membrane and suggests that the Mla pathway may have a role in anterograde phospholipid transport.
Proteins can be modified by multiple posttranslational modifications (PTMs), creating a PTM code that controls the function of proteins in space and time. Unraveling this complex PTM code is one of the great challenges in molecular biology. Here, using mass spectrometry-based assays, we focus on the most common PTMs-phosphorylation and O-GlcNAcylation-and investigate how they affect each other. We demonstrate two generic crosstalk mechanisms. First, we define a frequently occurring, very specific and stringent phosphorylation/ O-GlcNAcylation interplay motif, (pSp/T)P(V/A/T)(gS/gT), whereby phosphorylation strongly inhibits O-GlcNAcylation. Strikingly, this stringent motif is substantially enriched in the human (phospho)proteome, allowing us to predict hundreds of putative O-GlcNAc transferase (OGT) substrates. A set of these we investigate further and show them to be decent substrates of OGT, exhibiting a negative feedback loop when phosphorylated at the P-3 site. Second, we demonstrate that reciprocal crosstalk does not occur at PX(S/T)P sites, i.e., at sites phosphorylated by proline-directed kinases, which represent 40% of all sites in the vertebrate phosphoproteomes.O-GlcNAcylation | phosphorylation | crosstalk | signaling | regulation
We report unexpected mass spectrometric observations of glycosylated human leukocyte antigen (HLA) class I-bound peptides. Complemented by molecular modeling, in vitro enzymatic assays, and oxonium ion patterns, we propose that the observed O-linked glycans carrying up to five monosaccharides are extended O-GlcNAc’s rather than GalNAc-initiated O-glycans. A cytosolic O-GlcNAc modification is normally terminal and does not extend to produce a polysaccharide, but O-GlcNAc on an HLA peptide presents a special case because the loaded HLA class I complex traffics through the endoplasmic reticulum and Golgi apparatus on its way to the cell membrane, and is hence exposed to glycosyltransferases. In addition we report for the first time natural HLA class I presentation of O- and N-linked glycopeptides derived from membrane proteins. HLA class I peptides with centrally located oligosaccharides have been shown to be immunogenic and may therefore be important targets for immune-surveillance.
A wide variety of protein post-translational modifications (PTMs) decorate cellular proteins, regulating their structure, interactions and ultimately their function. The density of co-occurring PTMs on proteins can be very high, where multiple PTMs can positively or negatively influence each other's actions, termed PTM crosstalk. In this review, we highlight recent progress in the area of PTM crosstalk, whereby we focus on crosstalk between protein phosphorylation and O-GlcNAcylation. These two PTMs largely target identical (i.e., Ser and Thr) amino acids in proteins. Phosphorylation/O-GlcNAcylation crosstalk comes in many flavors, for instance by competition for the same site/residue (reciprocal crosstalk), as well as by modifications influencing each other in proximity or even distal on the protein sequence. PTM crosstalk is observed on the writers of these modifications (i.e., kinases and O-GlcNAc transferase), on the erasers (i.e., phosphatases and O-GlcNAcase), and on the readers and the substrates. We describe examples of all these different flavors of crosstalk, and additionally the methods that are emerging to better investigate in particular phosphorylation/O-GlcNAcylation crosstalk.
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