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A comprehensive pipeline for translational top-down proteomics from a single blood draw

Nature Protocols (2018) | Download Citation

Abstract

Top-down proteomics (TDP) by mass spectrometry (MS) is a technique by which intact proteins are analyzed. It has become increasingly popDesalting and concentrating GELFrEEular 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.

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Data availability

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).

Additional information

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.

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Acknowledgements

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.

Author information

Affiliations

  1. Departments of Chemistry and of Molecular Biosciences, Northwestern University, Evanston, IL, USA

    • Timothy K. Toby
    • , Luca Fornelli
    • , Kristina Srzentić
    •  & Neil L. Kelleher
  2. National Resource for Translational and Developmental Proteomics, Northwestern University, Evanston, IL, USA

    • Caroline J. DeHart
    •  & Neil L. Kelleher
  3. Comprehensive Transplant Center, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    • Josh Levitsky
    •  & John Friedewald

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Contributions

T.K.T. guided the development and application of the protocol at all stages. L.F. and K.S. provided technical expertise on UHPLC–MS/MS methods for TDMS and TDP. C.J.D. contributed greatly to the formalization of TD standard protocols for QC of TMDS and TDP experiments. J.L. and J.F. provided clinical and biorepository expertise, as well as access to the samples described in this protocol. N.L.K. supervised the work and provided guidance and direction on all components of the protocol.

Competing interests

N.L.K. declares an affiliation with Thermo Fisher Scientific. The remaining authors declare no competing interests.

Corresponding author

Correspondence to Neil L. Kelleher.

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https://doi.org/10.1038/s41596-018-0085-7

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