Proteoforms as the next proteomics currency Smith, Lloyd M; Kelleher, Neil L
Science (American Association for the Advancement of Science),
03/2018, Letnik:
359, Številka:
6380
Journal Article
Recenzirano
Odprti dostop
Identifying precise molecular forms of proteins can improve our understanding of function
Proteoforms—the different forms of proteins produced from the genome with a variety of sequence variations, ...splice isoforms, and myriad posttranslational modifications (
1
)—are critical elements in all biological systems (see the figure, left). Yang
et al.
(
2
) recently showed that the functions of proteins produced from splice variants from a given gene—different proteoforms—can be as different as those for proteins encoded by entirely different genes. Li
et al.
(
3
) showed that splice variants play a central role in modulating complex traits. However, the standard paradigm of proteomic analysis, the “bottom-up” strategy pioneered by Eng and Yates some 20 years ago (
4
), does not directly identify proteoforms. We argue that proteomic analysis needs to provide the identities and abundances of the proteoforms themselves, rather than just their peptide surrogates. Developing new proteome-wide strategies to accomplish this goal presents a formidable but not insurmountable technological challenge that will benefit the biomedical community.
Top Down proteomics: Facts and perspectives Catherman, Adam D.; Skinner, Owen S.; Kelleher, Neil L.
Biochemical and biophysical research communications,
03/2014, Letnik:
445, Številka:
4
Journal Article
Recenzirano
Odprti dostop
•Top Down versus Bottom Up proteomics analysis.•Separations methods for Top Down proteomics.•Developments in mass spectrometry instrumentation and fragmentation.•Native mass spectrometry.
The rise of ...the “Top Down” method in the field of mass spectrometry-based proteomics has ushered in a new age of promise and challenge for the characterization and identification of proteins. Injecting intact proteins into the mass spectrometer allows for better characterization of post-translational modifications and avoids several of the serious “inference” problems associated with peptide-based proteomics. However, successful implementation of a Top Down approach to endogenous or other biologically relevant samples often requires the use of one or more forms of separation prior to mass spectrometric analysis, which have only begun to mature for whole protein MS. Recent advances in instrumentation have been used in conjunction with new ion fragmentation using photons and electrons that allow for better (and often complete) protein characterization on cases simply not tractable even just a few years ago. Finally, the use of native electrospray mass spectrometry has shown great promise for the identification and characterization of whole protein complexes in the 100kDa to 1MDa regime, with prospects for complete compositional analysis for endogenous protein assemblies a viable goal over the coming few years.
From a molecular perspective, enactors of function in biology are intact proteins that can be variably modified at the genetic, transcriptional, or post-translational level. Over the past 30 years, ...mass spectrometry (MS) has become a powerful method for the analysis of proteomes. Prevailing bottom-up proteomics operates at the level of the peptide, leading to issues with protein inference, connectivity, and incomplete sequence modification information. Top-down proteomics (TDP), alternatively, applies MS at the proteoform level to analyze intact proteins with diverse sources of intramolecular complexity preserved during analysis. Fortunately, advances in prefractionation workflows, MS instrumentation, and dissociation methods for whole-protein ions have helped TDP emerge as an accessible and potentially disruptive modality with increasingly translational value. In this review, we discuss technical and conceptual advances in TDP, along with the growing power of proteoform-resolved measurements in clinical and translational research.
Proteomics has progressed radically in the last 5 years and is now on par with most genomic technologies in throughput and comprehensiveness. Analyzing peptide mixtures by liquid chromatography ...coupled to high-resolution mass spectrometry (LC-MS) has emerged as the main technology for in-depth proteome analysis whereas two-dimensional gel electrophoresis, low-resolution MALDI, and protein arrays are playing niche roles. MS-based proteomics is rapidly becoming quantitative through both label-free and stable isotope labeling technologies. The latest generation of mass spectrometers combines extremely high resolving power, mass accuracy, and very high sequencing speed in routine proteomic applications. Peptide fragmentation is mostly performed in low-resolution but very sensitive and fast linear ion traps. However, alternative fragmentation methods and high-resolution fragment analysis are becoming much more practical. Recent advances in computational proteomics are removing the data analysis bottleneck. Thus, in a few specialized laboratories, "precision proteomics" can now identify and quantify almost all fragmented peptide peaks. Huge challenges and opportunities remain in technology development for proteomics; thus, this is not "the beginning of the end" but surely "the end of the beginning."
Top-down mass spectrometry is an emerging technology which strives to preserve the post-translationally modified forms of proteins present in vivo by measuring them intact, rather than measuring ...peptides produced from them by proteolysis. The top-down technology is beginning to capture the interest of biologists and mass spectrometrists alike, with a main goal of deciphering interaction networks operative in cellular pathways. Here we outline recent approaches and applications of top-down mass spectrometry as well as an outlook for its future.
Genome mining has become a key technology to exploit natural product diversity. Although initially performed on a single-genome basis, the process is now being scaled up to mine entire genera, strain ...collections and microbiomes. However, no bioinformatic framework is currently available for effectively analyzing datasets of this size and complexity. In the present study, a streamlined computational workflow is provided, consisting of two new software tools: the 'biosynthetic gene similarity clustering and prospecting engine' (BiG-SCAPE), which facilitates fast and interactive sequence similarity network analysis of biosynthetic gene clusters and gene cluster families; and the 'core analysis of syntenic orthologues to prioritize natural product gene clusters' (CORASON), which elucidates phylogenetic relationships within and across these families. BiG-SCAPE is validated by correlating its output to metabolomic data across 363 actinobacterial strains and the discovery potential of CORASON is demonstrated by comprehensively mapping biosynthetic diversity across a range of detoxin/rimosamide-related gene cluster families, culminating in the characterization of seven detoxin analogues.
Covering: 2010 to 2021Organisms in nature have evolved into proficient synthetic chemists, utilizing specialized enzymatic machinery to biosynthesize an inspiring diversity of secondary metabolites. ...Often serving to boost competitive advantage for their producers, these secondary metabolites have widespread human impacts as antibiotics, anti-inflammatories, and antifungal drugs. The natural products discovery field has begun a shift away from traditional activity-guided approaches and is beginning to take advantage of increasingly available metabolomics and genomics datasets to explore undiscovered chemical space. Major strides have been made and now enable -omics-informed prioritization of chemical structures for discovery, including the prospect of confidently linking metabolites to their biosynthetic pathways. Over the last decade, more integrated strategies now provide researchers with pipelines for simultaneous identification of expressed secondary metabolites and their biosynthetic machinery. However, continuous collaboration by the natural products community will be required to optimize strategies for effective evaluation of natural product biosynthetic gene clusters to accelerate discovery efforts. Here, we provide an evaluative guide to scientific literature as it relates to studying natural product biosynthesis using genomics, metabolomics, and their integrated datasets. Particular emphasis is placed on the unique insights that can be gained from large-scale integrated strategies, and we provide source organism-specific considerations to evaluate the gaps in our current knowledge.
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•A useful reference for quantitative analysis of histone modifications.•Three general approaches to analyze histone modifications by mass spectrometry.•Analytical challenges of ...separation, instrumentation, and informatics for histones.•Strategies to implement LC–MS workflows for analysis of histone marks and codes.
Histones are a group of proteins with a high number of post-translational modifications, including methylation, acetylation, phosphorylation, and monoubiquitination, which play critical roles in every chromatin-templated activity. The quantitative analysis of these modifications using mass spectrometry (MS) has seen significant improvements over the last decade. It is now possible to perform large-scale surveys of dozens of histone marks and hundreds of their combinations on global chromatin. Here, we review the development of three MS strategies for analyzing histone modifications that have come to be known as Bottom Up, Middle Down, and Top Down. We also discuss challenges and innovative solutions for characterizing and quantifying complicated isobaric species arising from multiple modifications on the same histone molecule.
Top-down proteomics KELLEHER, Neil L
Analytical chemistry (Washington),
06/2004, Letnik:
76, Številka:
11
Journal Article
Recenzirano
Odprti dostop
A growing number of labs are using many types of mass spectrometers to directly analyze intact proteins and to improve conversion of mass spectrometry data into biological knowledge. Such approaches ...promise to determine molecular indicators of complex diseases and deepen our understanding of dynamic regulatory mechanisms in cell biology.
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