High-Resolution Native Mass Spectrometry Tamara, Sem; den Boer, Maurits A; Heck, Albert J. R
Chemical reviews,
04/2022, Letnik:
122, Številka:
8
Journal Article
Recenzirano
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Native mass spectrometry (MS) involves the analysis and characterization of macromolecules, predominantly intact proteins and protein complexes, whereby as much as possible the native structural ...features of the analytes are retained. As such, native MS enables the study of secondary, tertiary, and even quaternary structure of proteins and other biomolecules. Native MS represents a relatively recent addition to the analytical toolbox of mass spectrometry and has over the past decade experienced immense growth, especially in enhancing sensitivity and resolving power but also in ease of use. With the advent of dedicated mass analyzers, sample preparation and separation approaches, targeted fragmentation techniques, and software solutions, the number of practitioners and novel applications has risen in both academia and industry. This review focuses on recent developments, particularly in high-resolution native MS, describing applications in the structural analysis of protein assemblies, proteoform profiling ofamong othersbiopharmaceuticals and plasma proteins, and quantitative and qualitative analysis of protein–ligand interactions, with the latter covering lipid, drug, and carbohydrate molecules, to name a few.
Proteomics beyond trypsin Tsiatsiani, Liana; Heck, Albert J. R
The FEBS journal,
July 2015, Letnik:
282, Številka:
14
Journal Article
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Peptide‐centered shotgun analysis of proteins has been the core technology in mass spectrometry based proteomics and has enabled numerous biological discoveries, such as the large‐scale charting of ...protein–protein interaction networks, the quantitative analysis of protein post‐translational modifications and even the first drafts of the human proteome. The conversion of proteins into peptides in these so‐called bottom‐up approaches is nearly uniquely done by using trypsin as a proteolytic reagent. Here, we argue that our view of the proteome still remains incomplete and this is partially due to the nearly exclusive use of trypsin. Newly emerging alternative proteases and/or multi‐protease protein digestion aim to increase proteome sequence coverage and improve the identification of post‐translational modifications, through the analysis of complementary and often longer peptides, introducing an approach termed middle‐down proteomics. Of pivotal importance for this purpose is the identification of proteases beneficial for use in proteomics. Here, we describe some of the shortcomings of the nearly exclusive use of trypsin in proteomics and review the properties of other proteomics‐appropriate proteases. We describe favorable protease traits with an emphasis on middle‐down proteomics and suggest potential sources for the discovery of new proteases. We also highlight a few examples wherein the use of other proteases than trypsin enabled the generation of more comprehensive data sets leading to previously unexplored knowledge of the proteome.
ASPM (known as Asp in fly and ASPM-1 in worm) is a microcephaly-associated protein family that regulates spindle architecture, but the underlying mechanism is poorly understood. Here, we show that ...ASPM forms a complex with another protein linked to microcephaly, the microtubule-severing ATPase katanin. ASPM and katanin localize to spindle poles in a mutually dependent manner and regulate spindle flux. X-ray crystallography revealed that the heterodimer formed by the N- and C-terminal domains of the katanin subunits p60 and p80, respectively, binds conserved motifs in ASPM. Reconstitution experiments demonstrated that ASPM autonomously tracks growing microtubule minus ends and inhibits their growth, while katanin decorates and bends both ends of dynamic microtubules and potentiates the minus-end blocking activity of ASPM. ASPM also binds along microtubules, recruits katanin and promotes katanin-mediated severing of dynamic microtubules. We propose that the ASPM-katanin complex controls microtubule disassembly at spindle poles and that misregulation of this process can lead to microcephaly.
Native Mass Spectrometry: What is in the Name? Leney, Aneika C.; Heck, Albert J. R.
Journal of the American Society for Mass Spectrometry,
01/2017, Letnik:
28, Številka:
1
Journal Article
Recenzirano
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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 Abstract
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We demonstrate single-particle charge detection mass spectrometry on an Orbitrap for the analysis of megadalton biomolecular assemblies. We establish that the signal amplitudes of individual ions ...scale linearly with their charge, which can be used to resolve mixed ion populations, determine charge states and thus also determine the masses of individual ions. This enables the ultrasensitive analysis of heterogeneous protein assemblies including immunoglobulin oligomers, ribosomes, proteinaceous nanocontainers and genome-packed adeno-associated viruses.
Next-generation sequencing allows the analysis of genomes, including those representing disease states. However, the causes of most disorders are multifactorial, and systems-level approaches, ...including the analysis of proteomes, are required for a more comprehensive understanding. The proteome is extremely multifaceted owing to splicing and protein modifications, and this is further amplified by the interconnectivity of proteins into complexes and signalling networks that are highly divergent in time and space. Proteome analysis heavily relies on mass spectrometry (MS). MS-based proteomics is starting to mature and to deliver through a combination of developments in instrumentation, sample preparation and computational analysis. Here we describe this emerging next generation of proteomics and highlight recent applications.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Protein digestion using a dedicated protease represents a key element in a typical mass spectrometry (MS)-based shotgun proteomics experiment. Up to now, digestion has been predominantly performed ...with trypsin, mainly because of its high specificity, widespread availability and ease of use. Lately, it has become apparent that the sole use of trypsin in bottom-up proteomics may impose certain limits in our ability to grasp the full proteome, missing out particular sites of post-translational modifications, protein segments or even subsets of proteins. To overcome this problem, the proteomics community has begun to explore alternative proteases to complement trypsin. However, protocols, as well as expected results generated from these alternative proteases, have not been systematically documented. Therefore, here we provide an optimized protocol for six alternative proteases that have already shown promise in their applicability in proteomics, namely chymotrypsin, LysC, LysN, AspN, GluC and ArgC. This protocol is formulated to promote ease of use and robustness, which enable parallel digestion with each of the six tested proteases. We present data on protease availability and usage including recommendations for reagent preparation. We additionally describe the appropriate MS data analysis methods and the anticipated results in the case of the analysis of a single protein (BSA) and a more complex cellular lysate (Escherichia coli). The digestion protocol presented here is convenient and robust and can be completed in ∼2 d.
Adeno-associated viruses (AAVs) are increasingly used as gene therapy vectors. AAVs package their genome in a non-enveloped T = 1 icosahedral capsid of ~3.8 megaDalton, consisting of 60 subunits of 3 ...distinct viral proteins (VPs), which vary only in their N-terminus. While all three VPs play a role in cell-entry and transduction, their precise stoichiometry and structural organization in the capsid has remained elusive. Here we investigate the composition of several AAV serotypes by high-resolution native mass spectrometry. Our data reveal that the capsids assemble stochastically, leading to a highly heterogeneous population of capsids of variable composition, whereby even the single-most abundant VP stoichiometry represents only a small percentage of the total AAV population. We estimate that virtually every AAV capsid in a particular preparation has a unique composition. The systematic scoring of the simulations against experimental native MS data offers a sensitive new method to characterize these therapeutically important heterogeneous capsids.
Native mass spectrometry is an emerging technology that allows the topological investigation of intact protein complexes with high sensitivity and a theoretically unrestricted mass range. This unique ...tool provides complementary information to established technologies in structural biology, and also provides a link to high-throughput interactomics studies, which do not generate information on exact protein complex-composition, structure or dynamics. Here I review the current state of native mass spectrometry technology and discuss several important biological applications. I also describe current experimental challenges in native mass spectrometry, encouraging readers to contribute to solutions.
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