Oral Presentation 28th Annual Lorne Proteomics Symposium 2023

Enhanced proteoform characterisation using an integrated top-down and bottom-up mass spectrometry approach (#59)

Muhammad Zenaidee 1 , Ole Tietz 2 , Liming Hou 2 , Emilija Robinson 2 , Gene Hart-Smith 1
  1. Australian Proteome Analysis Facility, Macquarie University, Sydney, NSW, Australia
  2. Dementia Research Centre, Macquarie Medical School, Macquarie University, Sydney, NSW, Australia

The individual molecular forms of proteins – proteoforms – vastly outnumber genes. Since every proteoform holds potential to function in a unique capacity within the cell, effective proteoform characterisation methodologies will play a crucial role in driving systems-level understandings of cellular processes.

Despite this, the dominant analytical paradigm in proteomics, the bottom-up strategy, is generally ineffective when it comes to characterising individual proteoforms. This can be because the proteolytic peptides that are characterised in the bottom-up strategy are often shared across proteoforms, and/or individual modifications to the amino acid sequences are not measured. To circumvent this issue, top-down strategies that analyse entire proteoforms have been shown to be effective.1 However, these strategies generally require prior knowledge of the post-translational modifications, sequence variations, and other sources of molecular heterogeneity from which the proteoforms under analysis are derived, which can limit their utility.

In the present work, we describe a proteoform characterisation workflow that integrates the individual strengths of the top-down and bottom-up approaches, while overcoming their abovementioned limitations. Firstly, the workflow leverages the capacity for bottom-up proteomics to provide non-hypothesis driven discovery and localisation localise amino acid modifications. By subjecting samples containing multiple proteoforms to bottom-up analysis using Mascot error-tolerant sequence database searches, evidence for any chemical or post-translational modifications, amino acid substitutions or other protein modifications listed in the Unimod database are uncovered and localised to specific amino acids.2 Following this, the workflow employs top-down proteomics to measure the intact and fragment ion masses of the proteoforms under analysis. The fragment ions from the resulting data are assigned to both terminal and internal fragments using ClipsMS,3 with the moieties uncovered in the bottom-up analysis specified as variable modifications on the specific amino acids to which they were localised. The resulting information, together with the intact mass data, enables the co-existence of specific moieties on individual proteoforms, and thus each individual proteoform’s identity, to be thoroughly characterised.

We demonstrate this workflow using a sample of human full-length 2N4R tau (MAPT) proteoforms, subjected to bottom-up analysis on a Q-Exactive HF-X mass spectrometer and top-down EThcD analysis on a Tribrid Fusion Lumos mass spectrometer. Tau proteoforms derived from various combinations of cysteine to serine mutations are thoroughly characterised from the resulting data, providing proof-of-concept that our workflow can enable comprehensive proteoform characterisations without prior knowledge of the amino acid modifications or other alterations by which the proteoforms are defined.

  1. 1. Smith, L.M., Agar, J.N., Chamot-Rooke, J., Danis, P.O., Ge, Y., Loo, J.A., Paša-Tolić, L., Tsybin, Y.O., Kelleher, N.L. and Consortium for Top-Down Proteomics, The human proteoform project: defining the human proteome. Science Advances, 2021.
  2. 2. Creasy D.M., Cottrell J.S., Error tolerant searching of uninterpreted tandem mass spectrometry data. Proteomics. 2002.
  3. 3. Lantz C, Zenaidee M.A., Wei B, Hemminger Z., Ogorzalek Loo R.R., Loo J.A., ClipsMS: An Algorithm for Analyzing Internal Fragments Resulting from Top-Down Mass Spectrometry. Journal of Proteome Research. 2021.