Shining a spotlight on intact proteins

Abstract

PROTEOMICSVolume 14, Issue 10 p. 1125-1127 EditorialFree Access Shining a spotlight on intact proteins First published: 10 May 2014 https://doi.org/10.1002/pmic.201470073AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Ljiljana Paša-Tolić Christophe Masselon Cells react to cues from their environment using various mechanisms that include changes in metabolites, gene expression, protein binding partners, protein localization, and protein posttranslational modifications (PTMs), all of which contribute to altered cellular signatures that enable appropriate cellular responses. Given the seemingly infinite number of mechanisms available to affect protein function and modulate biological processes, the question arises as to how cells manage to interpret protein readouts to accomplish the appropriate cell-type specific response to a particular stimulus. Much of this complexity is originating at the protein rather than gene level. Accordingly, the term “proteoform” 1 was recently proposed to designate distinct protein products of a single gene, and account for the complexity arising from multiple sources, primarily PTMs and proteolytic processing (proteins), but also allelic variations (DNA) and alternative splicing (RNA). It is also becoming increasingly evident that different proteoforms, displaying specific patterns of PTMs, can prompt distinct cellular outcomes, with histone code as the most notable example. This protein heterogeneity is insidiously difficult to measure utilizing conventional peptide level (i.e., bottom-up) approaches. The bottom-up approach has undeniably made great strides in identifying, and in some cases quantifying, proteins and PTMs present in different biological contexts, but it is of limited value for accessing PTM codependency. The bottom-up approach is like the fable of the six blind men trying to describe an elephant. Each man's description is limited by the part of the elephant he can touch, and together the men's descriptions do not provide an accurate portrayal of an elephant. The bottom-up approach is inherently limited by inaccessibility of information and cannot account for the entirety of the proteoform. Given the limitations of bottom-up proteomics, there has been increasing interest in top-down proteome characterization strategies, where individual intact proteins are selected for analysis by tandem mass spectrometry (MS), without prior chemical or enzymatic proteolysis. Notably, top-down proteomics can potentially reveal information about coexisting PTMs and provide direct measurements of the relative abundance of different proteoforms, while it is very challenging to do so using bottom-up strategy. The field of top-down MS and proteomics has grown tremendously since the last PROTEOMICS Focus Issue on this topic appeared in October 2010. Much of this transformation can be attributed to technological advancements in chromatography, MS and bioinformatics. Top-down proteomics is clearly gaining momentum, as evidenced by the 25th ASMS Sanibel Conference on Top-down Proteomics in 2013, which brought together the broader top- down community, following the launch of the Consortium for Top Down Proteomics in 2012 (http://www.topdownproteomics.org/). The mission of the Consortium is “to promote innovative research, collaboration and education accelerating the comprehensive analysis of intact proteins.” In only a few years, that innovative research and collaboration is leading to a more complete understanding of the dynamic regulatory mechanisms in cell biology. While challenges persist, top-down proteomics is becoming recognized as an ideal tool for identifying the specific combination of PTMs occurring within the same proteoform and for measuring proteoform distributions to better understand complex signaling mechanisms that enable appropriate cellular responses to the internal and external cues. Accordingly, an update to top-down proteomics appears mandatory and we proudly offer the following collection of articles written by leading experts in the field to serve as a forum for presenting some of the latest strategies for characterization of intact proteins spanning applications from microbial virulence and human disease to protein structure, dynamics and function. We are grateful to all our colleagues who contributed their expertise by writing and/or reviewing articles, and for investing their time and sharing their valuable insights for this Special Issue. The articles collected in this issue address some of the long-standing challenges in the field, including different separation modalities coupled with advanced MS instrumentation for high-throughput characterization of (often chemically closely related) proteoforms 2-5; improved fragmentation methods to generate more complete sequence coverage 6, 7; integrated approaches that combine complementary information from bottom-up and top-down analyses 8; advanced computational/bioinformatics approaches to proteoform characterization 5, 9; tailored sample preparation protocols 10, 11; and emerging top-down MS imaging approaches 12, 13. Since not all sites on a protein are modified to the same extent under different (patho)physiological conditions, it is critical to explore specific PTM patterns associated with protein function alterations in response to a stimulus or a disease. 14, 15). The importance of biomarker based assays which measure the overall PTM status of a protein for diagnosing disease has been highlighted in several contributing articles. 14, 16, 17 Going a step further, it is critical to develop technologies to study how proteins interact to form cellular machines that perform essentially all cellular functions. 2, 18 These higher order protein complexes may act as information processing hubs determined by subunit composition and crosstalk between PTMs on multiple subunits, bringing additional level of complexity that we are yet to begin to address. We hope this Special Issue of PROTEOMICS on top-down proteomics provides a valuable resource for novices and seasoned practitioners of MS and proteomics, as well as biology, biochemistry and biomedicine. We also hope it stimulates further advances in the field. Enjoy the journey into the world of top-down proteomics. Ljiljana Paša-Tolić W.R. Wiley Environmental Molecular Sciences Laboratory Pacific Northwest National Laboratory, Richland, WA Christophe Masselon Institut de Recherches en Technologies et Sciences pour le Vivant (iRTSV) CEA/Grenoble References 1Smith, L. M., Kelleher, N. L., Consortium for Top Down Proteomics, Proteoform: a single term describing protein complexity. Nat. Methods, 2013, 10, 186– 187. CrossrefCASPubMedWeb of Science®Google Scholar 2Lakshmanan, R., Wolff, J. J., Alvarado, R., Loo, J. A., Top-down protein identification of proteasome proteins with nanoLC-FT-ICR-MS employing data-independent fragmentation methods. Proteomics 2014, 14, 1271– 1282. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 3Zhang, J., Corbett, J. R., Plymire, D. A., Greenberg, B. M., Patrie, S. M., Proteoform analysis of lipocalin-type prostaglandin D-synthase from human cerebrospinal fluid by isoelectric focusing and superficially porous liquid chromatography with fourier transform mass spectrometry. Proteomics 2014, 14, 1223– 1231. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 4Li, Y., Compton, P. D., Tran, J. C., Ntai, I., Kelleher, N. L., Optimizing capillary electrophoresis for top-down proteomics of 30–80 kDa proteins. Proteomics 2014, 14, 1158– 1164. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 5Dang, X., Scotcher, J., Wu, S., Chu, R. K. et al., The First Pilot Project of the Consortium for Top Down Proteomics: A status report. Proteomics 2014, 14, 1130– 1140. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 6Cannon, J. R., Kluwe, C., Ellington, A., Brodbelt, J. S., Characterization of green fluorescent proteins by 193 nm ultraviolet photodissociation mass spectrometry. Proteomics 2014, 14, 1165– 1173. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 7Bourgoin-Voillard, S., Leymarie, N., Costello, C. E., Top-down tandem mass spectrometry on RNase A and B using a Qh/FT-ICR hybrid mass spectrometer. Proteomics 2014, 14, 1174– 1184. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 8Dekker, L., Wu, S., Vanduijn, M., Tolić, N. et al., An integrated top-down and bottom-up proteomic approach to characterize the antigen-binding fragment of antibodies. Proteomics 2014, 14, 1239– 1248. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 9Jia, C., Yu, Q., Wang, J., Li, L., Qualitative and quantitative top-down mass spectral analysis of crustacean hyperglycemic hormones in response to feeding. Proteomics 2014, 14, 1185– 1194. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 10Auclair, J. R., Salisbury, J. P., Johnson, J. L., Petsko, G. A. et al., Artifacts to avoid while taking advantage of top-down mass spectrometry-based detection of protein S-thiolation. Proteomics 2014, 14, 1152– 1157. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 11Pan, J., Borchers, C. H., Top-down mass spectrometry and hydrogen/deuterium exchange for comprehensive structural characterization of interferons: Implications for biosimilars. Proteomics 2014, 14, 1249– 1258. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 12Kiss, A., Smith, D. F., Reschke, B. R., Powell, M. J., Heeren, R. M., Top-down mass spectrometry imaging of intact proteins by laser ablation ESI FT-ICR MS. Proteomics 2014, 14, 1283– 1289. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 13Ait-Belkacem, R., Berenguer, C., Villard, C., Ouafik, L. et al., MALDI imaging and in-source decay for top-down characterization of glioblastoma. Proteomics 2014, 14, 1290– 1301. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 14Gregorich, Z. R., Ge, Y., Top-down proteomics in health and disease: Challenges and opportunities. Proteomics 2014, 14, 1195– 1210. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 15Gault, J., Malosse, C., Machata, S., Millien, C. et al., Complete posttranslational modification mapping of pathogenic Neisseria meningitidis pilins requires top-down mass spectrometry. Proteomics 2014, 14, 1141– 1151. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 16Wu, S., Brown, J. N., Tolić, N., Meng, D. et al., Quantitative analysis of human salivary gland-derived intact proteome using top-down mass spectrometry. Proteomics 2014, 14, 1211– 1222. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 17Edwards, R. L., Griffiths, P., Bunch, J., Cooper, H. J., Compound heterozygotes and beta-thalassemia: Top-down mass spectrometry for detection of hemoglobinopathies. Proteomics 2014, 14, 1232– 1238. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 18Boeri-Erba, E., Investigating macromolecular complexes using top down mass spectrometry, Proteomics 2014, 14, 1259– 1270. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Volume14, Issue10Special Issue: Top-down ProteomicsMay 2014Pages 1125-1127 ReferencesRelatedInformation