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Methods in molecular exercise physiology
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Adam P. Sharples, Daniel C. Turner, Stephen Roth, Robert A. Seaborne, Brendan Egan, Mark Viggars, Jonathan C. Jarvis, Daniel J. Owens, Jatin G. Burniston, Piotr P. Gorski, Claire E. Stewart
The first proteomic studies of rodent (57) and human (58) muscle responses to exercise training used 2D gel electrophoresis to separate proteins into ‘proteoforms’ based on their isoelectric point and molecular weight. Proteoforms consist of different combinatorial patterns of splice variations and post-translational states of each protein and are the true functional entities of the proteome. However, 2D electrophoresis has limited power to resolve all proteins; for example, most studies in muscle report a few hundred proteins, which each might be observed as several different proteoforms (59). Most contemporary studies now use enzymatic digestion strategies that cleave proteins into peptides to offer more comprehensive coverage of the muscle proteome. Peptides are more soluble and, therefore, easier to handle than proteins and still contain enough information to infer the abundance, turnover rate or modification status of the parent proteins. Complex mixtures of peptides from muscle digests can be separated based on their relative hydrophobicity using reverse-phase liquid chromatography. This provides a reproducible means of delivering peptides to the mass spectrometer (see section directly below) over a defined period of time (60). Numerous different peptides may have a similar level of retention on the column and therefore elute into the mass spectrometer at the same time. Therefore, it is important to match the liquid chromatography separation with the speed and resolution of the mass spectrometer. If too many peptides are delivered to the mass spectrometer at any one point in time, information will be lost. Often, the very large numbers of peptides generated by digestion of whole tissues such as muscle necessitate prior fractionation of the sample in order to achieve the deepest levels of proteome coverage (55). Moreover, only a small proportion of the total number of copies of a protein carry post-translational modifications. Therefore, enrichment techniques are needed to study residue-specific covalent modifications, including phosphorylation (61), acetylation (62) and ubiquitination (53). Using these techniques, the connection between the complex combinatorial patterns of different modifications at different residues within each protein is lost, so these data do not provide insight at the proteoform level. Modern-day approaches to proteoform analysis include partial digestion strategies and sophisticated mass spectrometry of intact proteins but typically need to be targeted to individual proteins (63), so there is still a place for 2D gel electrophoresis, particularly in skeletal muscle where the majority of the myofibrillar proteome consists of closely related protein isoforms that exist as different splice variants or post-translational states, and have different responses to exercise stimuli (56).
How can platelet proteomics best be used to interrogate disease?
Published in Platelets, 2023
Although PTMs have been known for decades, the enormous complexity and importance of these regulatory protein changes have only gained more attention in proteome research in recent years. To give this extensive variety of protein variants of a protein a uniform name, the term proteoform was defined in 2013.9 Thus, the term proteoform includes variants of a protein based on variations or mutations in its gene or alternative splicing of its RNA. From highly sensitive shotgun analysis data it is currently estimated that platelets contain around 5000 different canonical proteins.10 Currently, there are approximately 400 recognized PTMs alongside other genetic and transcriptional variations.11 Thus, up to millions of different regulatory proteoforms can be present only in platelets.
Molecular insights into cancer drug resistance from a proteomics perspective
Published in Expert Review of Proteomics, 2019
Yao An, Li Zhou, Zhao Huang, Edouard C. Nice, Haiyuan Zhang, Canhua Huang
Depending on the application, proteomic methodologies are mainly divided into three approaches, namely top-down proteomics, middle down proteomics and bottom-up or shotgun proteomics [15–18]. Top-down proteomics (TDP) is the study at the proteoform level to analyze intact proteins with diverse sources of intramolecular complexity preserved during the analysis. It generally covers only relatively small proteins (3.5–30 kDa mass range). Bottom-up proteomics is the most common MS-based method, which is used for global peptide identification. In this method, a mixture of proteins is isolated and enzymatically or chemically cleaved into peptides followed by MS/MS analysis. Compared with TDP, bottom-up proteomics digests protein into peptides, leading to the existence of a gray zone between these proteomics approaches. This gap has been filled by the third approach, namely middle-down proteomics. This approach also uses protein digestion but aims to produce larger peptides than top-down proteomics, leading to better proteome coverage and increasing the potential to detect multiple PTMs, which is important for studying functionally relevant PTM crosstalk [16].
Origins and clinical relevance of proteoforms in pediatric malignancies
Published in Expert Review of Proteomics, 2019
Amanda Lorentzian, Anuli Uzozie, Philipp F. Lange
In addition to only moderate correlation of gene, transcript and protein level, variations on the whole genome and proteome level further contribute to this discordance. There are approximately 20,000 known protein-coding genes but over a hundred thousand protein proteoforms have been identified. Some speculate upwards of a million exist that have not yet been identified, eliminating the once popular idea that one gene encodes one protein [17,18]. There are several terms to describe these variants, such as ‘protein species’ or ‘isoforms’ [19,20]. However, the term ‘protein species’ does not differentiate between proteins that originated from the same gene or proteins from an entirely different gene and the term ‘isoforms’ specifically refers to different proteins arising from alternative splicing of a single gene. The term ‘proteoform’ applies to all the different proteins derived from a single gene, including all forms of genetic variation, alternative splicing, and post-translational modifications [21]. Groups of related proteoforms that are derived from a single gene and share a similar combination of modifications and variants are termed ‘proteoform families’ [17]. The existence of proteoforms creates molecularly distinct proteins that modulate a wide variety of biological processes including cell signaling, gene regulation, and activation [21]. Proteoforms can have distinct functions and can differ in their subcellular localization, binding partners, structure, and kinetics, among others. In-depth characterization has only been done for few of the known proteoforms and remains an important research topic [22].