Top-down proteomics (TDP) by mass spectrometry (MS) is a technique by which intact proteins are analyzed. It has become increasingly popular in translational research because of the value of characterizing distinct proteoforms of intact proteins. Compared to bottom-up proteomics (BUP) strategies, which measure digested peptide mixtures, TDP provides highly specific molecular information that avoids the bioinformatic challenge of protein inference. However, the technique has been difficult to implement widely because of inherent limitations of existing sample preparation methods and instrumentation. Recent improvements in proteoform pre-fractionation and the availability of high-resolution benchtop mass spectrometers have made it possible to use high-throughput TDP for the analysis of complex clinical samples. Here, we provide a comprehensive protocol for analysis of a common sample type in translational research: human peripheral blood mononuclear cells (PBMCs). The pipeline comprises multiple workflows that can be treated as modular by the reader and used for various applications. First, sample collection and cell preservation are described for two clinical biorepository storage schemes. Cell lysis and proteoform pre-fractionation by gel-eluted liquid fractionation entrapment electrophoresis are then described. Importantly, instrument setup and liquid chromatography–tandem MS are described for TDP analyses, which rely on high-resolution Fourier-transform MS. Finally, data processing and analysis are described using two different, application-dependent software tools: ProSight Lite for targeted analyses of one or a few proteoforms and TDPortal for high-throughput TDP in discovery mode. For a single sample, the minimum completion time of the entire experiment is 72 h.
Subscribe to Journal
Get full journal access for 1 year
only $41.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The .raw files converted to .mzML format used for this analysis are accessible at https://scholarworks.iu.edu/dspace/handle/2022/21091. In addition, the well-characterized proteoforms (C score >40) elucidated here are available for public access in the Consortium for Top Down Proteomics proteoform repository (http://atlas.topdownproteomics.org/proteoforms/?DataSetId=15).
Savaryn, J. P., Toby, T. K. & Kelleher, N. L. A researcher’s guide to mass spectrometry-based proteomics. Proteomics 16, 2435–2443 (2016).
Liumbruno, G., D’Alessandro, A., Grazzini, G. & Zolla, L. Blood-related proteomics. J. Proteomics 73, 483–507 (2010).
Zhu, P., Bowden, P., Zhang, D. & Marshall, J. G. Mass spectrometry of peptides and proteins from human blood. Mass Spectrom. Rev. 30, 685–732 (2011).
Zhang, Y. et al. Protein analysis by shotgun/bottom-up proteomics. Chem. Rev. 113, 2343–2394 (2013).
Nesvizhskii, A. I. & Aebersold, R. Interpretation of shotgun proteomic data: the protein inference problem. Mol. Cell. Proteomics 4, 1419–1440 (2005).
Savaryn, J. P. et al. The emergence of top-down proteomics in clinical research. Genome Med. 5, 53 (2013).
Smith, L. M. & Kelleher, N. L. Proteoform: a single term describing protein complexity. Nat. Methods 10, 186–187 (2013).
Compton, P. D., Zamdborg, L., Thomas, P. M. & Kelleher, N. L. On the scalability and requirements of whole protein mass spectrometry. Anal. Chem. 83, 6868–6874 (2011).
Toby, T. K., Fornelli, L. & Kelleher, N. L. Progress in top-down proteomics and the analysis of proteoforms. Annu. Rev. Anal. Chem. 9, 499–519 (2016).
LeDuc, R. D. et al. ProForma: a standard proteoform notation. J. Proteome Res. 17, 1321–1325 (2018).
Bunger, M. K. et al. Automated proteomics of E. coli via top-down electron-transfer dissociation mass spectrometry. Anal. Chem. 80, 1459–1467 (2008).
Li, Y. et al. Optimizing capillary electrophoresis for top-down proteomics of 30-80 kDa proteins. Proteomics 14, 1158–1164 (2014).
Ferguson, J. T., Wenger, C. D., Metcalf, W. W. & Kelleher, N. L. Top-down proteomics reveals novel protein forms expressed in Methanosarcina acetivorans. J. Am. Soc. Mass Spectrom. 20, 1743–1750 (2009).
Kellie, J. F. et al. Robust analysis of the yeast proteome under 50 kDa by molecular-mass-based fractionation and top-down mass spectrometry. Anal. Chem. 84, 209–215 (2012).
Meng, F. et al. Processing complex mixtures of intact proteins for direct analysis by mass spectrometry. Anal. Chem. 74, 2923–2929 (2002).
Meng, F. et al. Molecular-level description of proteins from Saccharomyces cerevisiae using quadrupole FT hybrid mass spectrometry for top down proteomics. Anal. Chem. 76, 2852–2858 (2004).
Ntai, I. et al. Applying label-free quantitation to top down proteomics. Anal. Chem. 86, 4961–4968 (2014).
Tran, J. C. & Doucette, A. A. Gel-eluted liquid fraction entrapment electrophoresis: an electrophoretic method for broad molecular weight range proteome separation. Anal. Chem. 80, 1568–1573 (2008).
Tran, J. C. & Doucette, A. A. Multiplexed size separation of intact proteins in solution phase for mass spectrometry. Anal. Chem. 81, 6201–6209 (2009).
Hardman, M. & Makarov, A. A. Interfacing the orbitrap mass analyzer to an electrospray ion source. Anal. Chem. 75, 1699–1705 (2003).
Hu, Q. et al. The Orbitrap: a new mass spectrometer. J. Mass Spectrom. 40, 430–443 (2005).
Michalski, A. et al. Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer. Mol. Cell. Proteomics 10, M111.011015 (2011).
Michalski, A. et al. Ultra high resolution linear ion trap Orbitrap mass spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and versatile peptide fragmentation modes. Mol. Cell. Proteomics 11, O111.013698 (2012).
Olsen, J. V. et al. A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed. Mol. Cell. Proteomics 8, 2759–2769 (2009).
Senko, M. W. et al. Novel parallelized quadrupole/linear ion trap/Orbitrap tribrid mass spectrometer improving proteome coverage and peptide identification rates. Anal. Chem. 85, 11710–11714 (2013).
Ahlf, D. R. et al. Evaluation of the compact high-field orbitrap for top-down proteomics of human cells. J. Proteome Res. 11, 4308–4314 (2012).
Cai, W. et al. MASH Suite Pro: a comprehensive software tool for top-down proteomics. Mol. Cell. Proteomics 15, 703–714 (2016).
Guner, H. et al. MASH Suite: a user-friendly and versatile software interface for high-resolution mass spectrometry data interpretation and visualization. J. Am. Soc. Mass Spectrom. 25, 464–470 (2014).
Kou, Q., Xun, L. & Liu, X. TopPIC: a software tool for top-down mass spectrometry-based proteoform identification and characterization. Bioinformatics 32, 3495-3497 (2016).
Leduc, R. D. & Kelleher, N. L. Using ProSight PTM and related tools for targeted protein identification and characterization with high mass accuracy tandem MS data. Curr. Protoc. Bioinformatics Chapter 13:Unit 13.6 (2007).
LeDuc, R. D. et al. ProSight PTM: an integrated environment for protein identification and characterization by top-down mass spectrometry. Nucleic Acids Res. 32, W340–W345 (2004).
Liu, X. et al. Protein identification using top-down. Mol. Cell. Proteomics 11, M111.008524 (2012).
Taylor, G. K. et al. Web and database software for identification of intact proteins using “top down” mass spectrometry. Anal. Chem. 75, 4081–4086 (2003).
Zamdborg, L. et al. ProSight PTM 2.0: improved protein identification and characterization for top down mass spectrometry. Nucleic Acids Res. 35, W701–W706 (2007).
Kellie, J. F. et al. Quantitative measurement of intact alpha-synuclein proteoforms from post-mortem control and Parkinson’s disease brain tissue by intact protein mass spectrometry. Sci. Rep. 4, 5797 (2014).
Laouirem, S. et al. Progression from cirrhosis to cancer is associated with early ubiquitin post-translational modifications: identification of new biomarkers of cirrhosis at risk of malignancy. J. Pathol. 234, 452–463 (2014).
Martelli, C. et al. Integrated proteomic platforms for the comparative characterization of medulloblastoma and pilocytic astrocytoma pediatric brain tumors: a preliminary study. Mol. Biosyst. 11, 1668–1683 (2015).
Zhang, J. et al. Top-down quantitative proteomics identified phosphorylation of cardiac troponin I as a candidate biomarker for chronic heart failure. J. Proteome Res. 10, 4054–4065 (2011).
Desiderio, C. et al. Cerebrospinal fluid top-down proteomics evidenced the potential biomarker role of LVV- and VV-hemorphin-7 in posterior cranial fossa pediatric brain tumors. Proteomics 12, 2158–2166 (2012).
Cabras, T. et al. Significant modifications of the salivary proteome potentially associated with complications of Down syndrome revealed by top-down proteomics. Mol. Cell. Proteomics 12, 1844–1852 (2013).
Iavarone, F. et al. Characterization of salivary proteins of schizophrenic and bipolar disorder patients by top-down proteomics. J. Proteomics 103, 15–22 (2014).
De Petris, L. et al. Tumor expression of S100A6 correlates with survival of patients with stage I non-small-cell lung cancer. Lung Cancer 63, 410–417 (2009).
Florell, S. R. et al. Preservation of RNA for functional genomic studies: a multidisciplinary tumor bank protocol. Mod. Pathol. 14, 116–128 (2001).
Savaryn, J. P. et al. Comparative top down proteomics of peripheral blood mononuclear cells from kidney transplant recipients with normal kidney biopsies or acute rejection. Proteomics 16, 2048–2058 (2016).
Toby, T. K. et al. Proteoforms in peripheral blood mononuclear cells as novel rejection biomarkers in liver transplant recipients. Am. J. Transplant. 17, 2458–2467 (2017).
DeHart, C. J. et al. Bioinformatics analysis of top-down mass spectrometry data with ProSight Lite. Methods Mol. Biol. 1558, 381–394 (2017).
Fellers, R. T. et al. ProSight Lite: graphical software to analyze top-down mass spectrometry data. Proteomics 15, 1235–1238 (2015).
LeDuc, R. D. et al. The C-score: a Bayesian framework to sharply improve proteoform scoring in high-throughput top down proteomics. J. Proteome Res. 13, 3231–3240 (2014).
Rader, J. S. et al. A unified sample preparation protocol for proteomic and genomic profiling of cervical swabs to identify biomarkers for cervical cancer screening. Proteomics Clin. Appl. 2, 1658–1669 (2008).
Anderson, L. C. et al. Identification and characterization of human proteoforms by top-down LC-21 Tesla FT-ICR mass spectrometry. J. Proteome Res. 16, 1087–1096 (2017).
Ntai, I., Toby, T. K., LeDuc, R. D. & Kelleher, N. L. A method for label-free, differential top-down proteomics. Methods Mol. Biol. 1410, 121–133 (2016).
Denisov, E., Damoc, E., Lange, O. & Makarov, A. Orbitrap mass spectrometry with resolving powers above 1,000,000. Int. J. Mass Spectrom. 325, 80–85 (2012).
Olsen, J. V. et al. Higher-energy C-trap dissociation for peptide modification analysis. Nat. Methods 4, 709–712 (2007).
Durbin, K. R. et al. Quantitation and identification of thousands of human proteoforms below 30 kDa. J. Proteome Res. 15, 976–982 (2016).
Afgan, E. et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Res. 44, W3–W10 (2016).
Giardine, B. et al. Galaxy: a platform for interactive large-scale genome analysis. Genome Res. 15, 1451–1455 (2005).
Liu, X. et al. Deconvolution and database search of complex tandem mass spectra of intact proteins: a combinatorial approach. Mol. Cell. Proteomics 9, 2772–2782 (2010).
Carvalho, P. C. et al. YADA: a tool for taking the most out of high-resolution spectra. Bioinformatics 25, (2734–2736 (2009).
Meng, F. et al. Informatics and multiplexing of intact protein identification in bacteria and the archaea. Nat. Biotechnol. 19, 952–957 (2001).
Toby, T. K. et al. Proteoforms in peripheral blood mononuclear cells as novel rejection biomarkers in liver transplant recipients. Am. J. Transplant. 17, 2458-2467 (2017).
Kessner, D. et al. ProteoWizard: open source software for rapid proteomics tools development. Bioinformatics 24, 2534–2536 (2008).
Higdon, R., Haynes, W. & Kolker, E. Meta-analysis for protein identification: a case study on yeast data. OMICS 14, 309–314 (2010).
Fornelli, L. et al. Analysis of intact monoclonal antibody IgG1 by electron transfer dissociation Orbitrap FTMS. Mol. Cell. Proteomics 11, 1758–1767 (2012).
Fornelli, L. et al. Advancing top-down analysis of the human proteome using a benchtop quadrupole-Orbitrap mass spectrometer. J. Proteome Res. 16, 609–618 (2017).
Durbin, K. R. et al. Autopilot: an online data acquisition control system for the enhanced high-throughput characterization of intact proteins. Anal. Chem. 86, 1485–1492 (2014).
Richards, A. L. et al. One-hour proteome analysis in yeast. Nat. Protoc. 10, 701–714 (2015).
Fornelli, L. et al. Top-down analysis of immunoglobulin G isotypes 1 and 2 with electron transfer dissociation on a high-field Orbitrap mass spectrometer. J. Proteomics 159, 67-76 (2017).
Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996).
Wessel, D. & Flugge, U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141–143 (1984).
We thank the following members of the Kelleher Research Group and Proteomics Center of Excellence for helpful discussions and experimental assistance: R. Fellers, J. Greer, P. Compton, and P. Thomas. We also acknowledge the Northwestern Comprehensive Transplant Center Biorepository Core. This work was supported by the Paul G. Allen Family Foundation (grant award 11715 to N.L.K.), and the National Institutes of Health via the National Resource for Translational and Developmental Proteomics under grant P41 GM108569 from the National Institute of General Medical Sciences. T.K.T. was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award no. T32GM105538, as well as by an American Chemical Society Division of Analytical Chemistry Fellowship, sponsored by the Society for Analytical Chemists of Pittsburgh.
N.L.K. declares an affiliation with Thermo Fisher Scientific. The remaining authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key references using this protocol
Savaryn, J. P. et al. Proteomics 16, 2048–2058 (2016): https://onlinelibrary.wiley.com/doi/abs/10.1002/pmic.201600008
Toby, T. K. et al. Am. J. Transplant. 17, 2458–2467 (2017): https://onlinelibrary.wiley.com/doi/full/10.1111/ajt.14359
ProSight Lite, TDPortal, and Top Down Viewer are freely available online at a website hosted by the National Resource for Translational and Developmental Proteomics: http://nrtdp.northwestern.edu/resource-software.
About this article
Cite this article
Toby, T.K., Fornelli, L., Srzentić, K. et al. A comprehensive pipeline for translational top-down proteomics from a single blood draw. Nat Protoc 14, 119–152 (2019). https://doi.org/10.1038/s41596-018-0085-7
Analytical Chemistry (2020)
Nature Protocols (2020)
Briefings in Bioinformatics (2020)
Molecular & Cellular Proteomics (2020)
Constructing Human Proteoform Families Using Intact-Mass and Top-Down Proteomics with a Multi-Protease Global Post-Translational Modification Discovery Database
Journal of Proteome Research (2019)