Proteomics beyond trypsin Tsiatsiani, Liana; Heck, Albert J. R
The FEBS journal,
July 2015, Volume:
282, Issue:
14
Journal Article
Peer reviewed
Open access
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.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Protein post‐translational modifications (PTMs) allow the cell to regulate protein activity and play a crucial role in the response to changes in external conditions or internal states. Advances in ...mass spectrometry now enable proteome wide characterization of PTMs and have revealed a broad functional role for a range of different types of modifications. Here we review advances in the study of the evolution and function of PTMs that were spurred by these technological improvements. We provide an overview of studies focusing on the origin and evolution of regulatory enzymes as well as the evolutionary dynamics of modification sites. Finally, we discuss different mechanisms of altering protein activity via post‐translational regulation and progress made in the large‐scale functional characterization of PTM function.
Advances in proteomics have opened new avenues for the analysis of the evolution of protein post‐translational modifications (PTMs) and have enabled the large‐scale functional characterization of a range of different modifications types.
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FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
Microtubules (MTs) are an essential component of the neuronal cytoskeleton; they are involved in various aspects of neuron development, maintenance, and functions including polarization, synaptic ...plasticity, and transport. Neuronal MTs are highly heterogeneous due to the presence of multiple tubulin isotypes and extensive post‐translational modifications (PTMs). These PTMs—most notably detyrosination, acetylation, and polyglutamylation—have emerged as important regulators of the neuronal microtubule cytoskeleton. With this review, we summarize what is currently known about the impact of tubulin PTMs on microtubule dynamics, neuronal differentiation, plasticity, and transport as well as on brain function in normal and pathological conditions, in particular during neuro‐degeneration. The main therapeutic approaches to neuro‐diseases based on the modulation of tubulin PTMs are also summarized. Overall, the review indicates how tubulin PTMs can generate a large number of functionally specialized microtubule sub‐networks, each of which is crucial to specific neuronal features.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Breast cancer is a fatal cancer with the highest mortality in female. New strategies for anti-breast cancer are still urgently needed. Catalpol, an iridoid glycoside extracted from the traditional ...Chinese medicinal plant Rehmannia glutinosa, has shown anticancer efficacy in various cancer cells. However, its effect on breast cancer remains unclear. In this study, we aim to investigate the anti-breast cancer activity of catalpol and elucidate its underlying mechanism. Cell counting kit-8 (CCK-8) and morphology change showed that catalpol could inhibit the proliferation and viability of MCF-7 cells. Catalpol administration reduced the tumor volume in xenograft model. Catalpol induced apoptosis in MCF-7 cells confirmed by Hoechst 33342 staining and Annexin V-FITC/PI double staining. In vivo, catalpol also induced apoptosis as seen from the increased level of terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) in tumor. According to JC-1 and Dichlorodi-hydrofluorescein Diacetate (DCFH-DA) staining, loss of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) generation was found in MCF-7 cells treated with catalpol. Furthermore, catalpol also increased the level of cytoplasmic cytochrome c and activity of caspase-3 in MCF-7 cells. Likewise, histopathological and immunohistochemical (IHC) assay also found that catalpol enhanced the levels of cytochrome c and caspase-3 in breast cancer tissues. Ultimately, acetylation, 2-hydroxyisobutyrylation and lactylation were dramatically increased, whereas succinylation, malonylation and phosphorylation were markedly decreased in the breast cancer tumor treated with catalpol. Taken together, catalpol inhibited breast cancer in vitro and in vivo through induction of apoptosis via mitochondria apoptosis pathway and regulation of protein post-translational modifications (PTMs). Thus, it can be considered as an excellent candidate compound for treatment of breast cancer.
•Catalpol inhibited the growth of MCF-7 cells in vitro and in vivo.•Catalpol promoted the apoptosis in vitro and in vivo.•Catalpol triggered the mitochondria apoptosis pathway in vitro and in vivo.•Catalpol intervened PTMs in MCF-7 tumor in vivo.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The emergence of proteomics has led to major technological advances in mass spectrometry (MS). These advancements not only benefitted MS‐based high‐throughput proteomics but also increased the impact ...of mass spectrometry on the field of structural and molecular biology. Here, we review how state‐of‐the‐art MS methods, including native MS, top‐down protein sequencing, cross‐linking‐MS, and hydrogen–deuterium exchange‐MS, nowadays enable the characterization of biomolecular structures, functions, and interactions. In particular, we focus on the role of mass spectrometry in integrated structural and molecular biology investigations of biological macromolecular complexes and cellular machineries, highlighting work on CRISPR–Cas systems and eukaryotic transcription complexes.
Starting from a historical perspective, Albert Heck and colleagues showcase how state‐of‐the‐art mass spectrometry approaches have evolved into powerful tools to elucidate structure and function of proteins and protein complexes.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Byonic is the name of a software package for peptide and protein identification by tandem mass spectrometry. This software, which has only recently become commercially available, facilitates a much ...wider range of search possibilities than previous search software such as SEQUEST and Mascot. Byonic allows the user to define an essentially unlimited number of variable modification types. Byonic also allows the user to set a separate limit on the number of occurrences of each modification type, so that a search may consider only one or two chance modifications such as oxidations and deamidations per peptide, yet allow three or four biological modifications such as phosphorylations, which tend to cluster together. Hence, Byonic can search for tens or even hundreds of modification types simultaneously without a prohibitively large combinatorial explosion. Byonic's Wildcard Search allows the user to search for unanticipated or even unknown modifications alongside known modifications. Finally, Byonic's Glycopeptide Search allows the user to identify glycopeptides without prior knowledge of glycan masses or glycosylation sites.
α-enolase (ENOA) is a metabolic enzyme involved in the synthesis of pyruvate. It also acts as a plasminogen receptor and thus mediates activation of plasmin and extracellular matrix degradation. In ...tumor cells, ΕΝΟΑ is upregulated and supports anaerobic proliferation (Warburg effect), it is expressed at the cell surface, where it promotes cancer invasion, and is subjected to a specific array of post-translational modifications, namely acetylation, methylation and phosphorylation. Both ENOA overexpression and its post-translational modifications could be of diagnostic and prognostic value in cancer. This review will discuss recent information on the biochemical, proteomics and immunological characterization of ENOA, particularly its ability to trigger a specific humoral and cellular immune response. In our opinion, this information can pave the way for effective new therapeutic and diagnostic strategies to counteract the growth of the most aggressive human disease.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
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