title: Toward an Optimized Workflow for Middle-Down Proteomics.
abstract: 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 < Mw< 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.
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research, Utrecht University , Padualaan 8, 3584 CH Utrecht, The Netherlands.|Netherlands Proteomics Center , Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Alba
authors.initials: A
authors.lastname: Cristobal
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research, Utrecht University , Padualaan 8, 3584 CH Utrecht, The Netherlands.|Netherlands Proteomics Center , Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Fabio
authors.initials: F
authors.lastname: Marino
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research, Utrecht University , Padualaan 8, 3584 CH Utrecht, The Netherlands.|Netherlands Proteomics Center , Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Harm
authors.initials: H
authors.lastname: Post
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research, Utrecht University , Padualaan 8, 3584 CH Utrecht, The Netherlands.|Netherlands Proteomics Center , Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Henk W P
authors.initials: HW
authors.lastname: van den Toorn
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research, Utrecht University , Padualaan 8, 3584 CH Utrecht, The Netherlands.|Netherlands Proteomics Center , Padualaan 8, 3584 CH Utrecht, The Netherlands.|Departments of Chemistry and Biochemistry, University of Oxford , New Biochemistry Building, South Parks Road, Oxford, OX1 3QU Oxfordshire, United Kingdom.
authors.forename: Shabaz
authors.identifier: 0000-0003-2640-9560
authors.initials: S
authors.lastname: Mohammed
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research, Utrecht University , Padualaan 8, 3584 CH Utrecht, The Netherlands.|Netherlands Proteomics Center , Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Albert J R
authors.identifier: 0000-0002-2405-4404
authors.initials: AJ
authors.lastname: Heck
mesh_terms: D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D010316:Particle Size; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics
chemical_list: D010455:Peptides; D010447:Peptide Hydrolases