4.8 Article

Toward an Optimized Workflow for Middle-Down Proteomics

期刊

ANALYTICAL CHEMISTRY
卷 89, 期 6, 页码 3318-3325

出版社

AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.6b03756

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资金

  1. Netherlands Organisation for Scientific Research (NWO) via the Roadmap Initiative Proteins Work [184.032.201]
  2. VIDI grant [700.10.429]
  3. TOP-Punt Grant [718.015.003]
  4. European Union Horizon programme FET-OPEN project MSmed [686547]

向作者/读者索取更多资源

Mass spectrometry (MS)-based proteomics workflows can crudely be classified into two distinct regimes, targeting either relatively small peptides (i.e., 0.7 kDa < MW < 3.0 kDa) or small to medium sized intact proteins (i.e., 10 kDa < M-w < 30 kDa), respectively, termed bottom-up and top down proteomics. Recently, a niche has started to be explored covering the analysis of middle-range peptides (i.e., 3.0 kDa < MW < 10 kDa), aptly termed middle-down proteomics. Although middle-down proteomics can follow, in principle, a modular workflow similar to that of bottom-up proteomics, we hypothesized that each of these modules would benefit from targeted optimization to improve its overall performance in the analysis of middle-range sized peptides. Hence, to generate middle-range sized peptides from cellular lysates, we explored the use of the proteases Asp-N and Glu-C and a nonenzymatic acid induced cleavage. To increase the depth of the proteome, a strong cation exchange (SCX) separation, carefully tuned to improve the separation of longer peptides, combined with reversed phase-liquid chromatography (RP-LC) using columns packed with material possessing a larger pore size, was used. Finally, after evaluating the combination of potentially beneficial MS settings, we also assessed the peptide fragmentation techniques, including higher-energy collision dissociation (HCD), electron-transfer dissociation (ETD), and electron-transfer combined with higher-energy collision dissociation (EThcD), for characterization of middle-range sized peptides. These combined improvements clearly improve the detection and sequence coverage of middle range peptides and should guide researchers to explore further how middle-down proteomics may lead to an improved proteome coverage, beneficial for, among other things, the enhanced analysis of (co-occurring) post-translational modifications.

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