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Toward an Optimized Workflow for Middle-Down Proteomics
Open accessAbstract
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.
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Cites
Eng, J. K., McCormack, A. L., & Yates, J. R. (1994) · Journal of the American Society for Mass Spectrometry · doi:10.1016/1044-0305(94)80016-2 · Cited by 6,484
2016 update of the PRIDE database and its related tools
Vizcaíno, J. A., Csordás, A., del‐Toro, N., Dianes, J. A., Griss, J., Lavidas, I., Mayer, G., Pérez‐Riverol, Y., Reisinger, F., Ternent, T., et al. (2015) · Nucleic Acids Research · doi:10.1093/nar/gkv1145 · Cited by 3,466
Semi-supervised learning for peptide identification from shotgun proteomics datasets
Käll, L., Canterbury, J. D., Weston, J., Noble, W. S., & MacCoss, M. J. (2007) · Nature Methods · doi:10.1038/nmeth1113 · Cited by 2,424
Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry
Syka, J. E. P., Coon, J. J., Schroeder, M. J., Shabanowitz, J., & Hunt, D. F. (2004) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0402700101 · Cited by 2,370
Proteoform: a single term describing protein complexity
The Consortium for Top Down Proteomics, Smith, L. M., & Kelleher, N. L. (2013) · Nature Methods · doi:10.1038/nmeth.2369 · Cited by 1,528
Protein Analysis by Shotgun/Bottom-up Proteomics
Zhang, Y., Fonslow, B. R., Shan, B., Baek, M.-C., & Yates, J. R. (2013) · Chemical Reviews · doi:10.1021/cr3003533 · Cited by 1,491
Interpretation of Shotgun Proteomic Data
Nesvizhskii, A. I., & Aebersold, R. (2005) · Molecular & Cellular Proteomics · doi:10.1074/mcp.r500012-mcp200 · Cited by 1,025
Deep proteome and transcriptome mapping of a human cancer cell line
Nagaraj, N., Wisniewski, J. R., Geiger, T., Cox, J., Kircher, M., Kelso, J., Pääbo, S., & Mann, M. (2011) · Molecular Systems Biology · doi:10.1038/msb.2011.81 · Cited by 979
Mapping intact protein isoforms in discovery mode using top-down proteomics
Tran, J. C., Zamdborg, L., Ahlf, D. R., Lee, J. E., Catherman, A. D., Durbin, K. R., Tipton, J. D., Vellaichamy, A., Kellie, J. F., Li, M., et al. (2011) · Nature · doi:10.1038/nature10575 · Cited by 665
Shotgun identification of protein modifications from protein complexes and lens tissue
MacCoss, M. J., McDonald, W. H., Saraf, A., Sadygov, R. G., Clark, J., Tasto, J. J., Gould, K. L., Wolters, D. A., Washburn, M. P., Weiss, A., et al. (2002) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.122231399 · Cited by 593
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Cumming, M. G., & Biggar, K. K. (2025) · Biomedicines · doi:10.3390/biomedicines13112825 · Cited by 3
Middle-down proteomics: the pursuit for longer peptides
Hovey, O. F. J., Lajoie, G. A., & Cooper, T. T. (2025) · Expert Review of Proteomics · doi:10.1080/14789450.2025.2604157 · Cited by 3
Ion Activation Methods for Top‐Down Proteomics
Walker, J. N., & Brodbelt, J. S. (2026) · Mass Spectrometry Reviews · doi:10.1002/mas.70023 · Cited by 4
Hamza, G. M., Miele, E., Wojchowski, D. M., Toran, P. T., Worsfold, C. R., Anthonymuthu, T. S., Bergo, V. B., Zhang, A. X., & Silva, J. C. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms241210060 · Cited by 2
Takemori, A., Kaulich, P. T., Tholey, A., & Takemori, N. (2025) · PROTEOMICS · doi:10.1002/pmic.70007 · Cited by 2
Mass Spectrometry-Based Proteomics and Metaproteomics Analysis of Ancient Manuscripts
Creydt, M., & Fischer, M. J. (2021) · De Gruyter eBooks · doi:10.1515/9783110753301-010 · Cited by 2
Panomics—Fingerprinting Approaches for Food Fraud Detection
Creydt, M., & Fischer, M. J. (2023) · Elsevier eBooks · doi:10.1016/b978-0-12-822521-9.00176-3 · Cited by 2
Yang, L., Stanisheuski, S., Song, Z., Bracha, S., & Maier, C. S. (2023) · European Journal of Mass Spectrometry · doi:10.1177/14690667231202766 · Cited by 1
Soo, P., Lee, C.-C., Shie, M.-F., Patil, A. A., Descanzo, M. J. N., Chin, Y.-C., Chen, H.-A., Horng, Y., Lin, C., Lee, J., et al. (2024) · Analytical Methods · doi:10.1039/d4ay00314d · Cited by 1
Declerck, K., Savić, N. D., Parammal, M. J., Seno, C., Bruylants, G., Roo, J. D., & Parac‐Vogt, T. N. (2025) · Angewandte Chemie · doi:10.1002/ange.202512482 · Cited by 1
Xiong, X., Qingge, L., Zhu, B., & Liu, X. (2026) · Analytical Chemistry · doi:10.1021/acs.analchem.5c06097 · Cited by 1
Kazieva, L. S., Farafonova, T. E., & Zgoda, V. G. (2023) · Biomeditsinskaya Khimiya · doi:10.18097/pbmc20236901005 · Cited by 1
Confirmation de biomarqueurs pour le pronostic du sepsis et développement de tests rapides
Dubois, C. (2019) · theses.fr (ABES) · doi:10.70675/4101c43dzc2b3z4ae2zb315z16d8d8252081 · Cited by 1
Galluzzi, F. (2021) · theses.fr (ABES) · doi:10.70675/544c95bfz2ddbz4ecaza12dz64e75d212990 · Cited by 1
Data Trends in Protein Analysis for Safety Assessments
Wang, R., Hill, R. C., & Houston, N. L. (2019) · ACS symposium series · doi:10.1021/bk-2019-1300.ch005
Thuy-Boun, P. S., Wang, A. Y., Crissien-Martinez, A., Xu, J. H., Chatterjee, S., Stupp, G. S., Su, A. I., Coyle, W., & Wolan, D. W. (2020) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2020.11.11.378836
Diving into cellular signalling
Leijten, N. M. (2021) · Utrecht University Repository (Utrecht University) · doi:10.33540/622
Una compleja red política y empresarial: La familia López, 1889-1972
Marco, J. M. U. (2001) · Dialnet (Universidad de la Rioja)
Warren, P. D., & Smith, M. H. (2023) · Journal of Molecular Recognition · doi:10.1002/jmr.3011
Mathew, A. (2023) · Research Publications (Maastricht University) · doi:10.26481/dis.20231017am
Bioinformatics in Green and Sustainable Technologies
Rathod, S., Patel, N. A., & Prajapati, B. G. (2024) · Nanotechnology and nanomaterials. · doi:10.5772/intechopen.112108 · Cited by 1
Rigante, E. C. L., Calvano, C. D., Losito, I., & Cataldi, T. R. I. (2025) · Talanta · doi:10.1016/j.talanta.2025.128352
Liu, R., Liang, Z., Xia, S., Luo, Y., & Li, H. (2026) · Analytica Chimica Acta · doi:10.1016/j.aca.2026.345161
Gao, R., Anthony, T. E., de Azambuja, F., Doonan, C. J., & Parac‐Vogt, T. N. (2026) · ACS Applied Materials & Interfaces · doi:10.1021/acsami.5c25083
Pythoud, N. (2020) · theses.fr (ABES) · doi:10.70675/659636cez02e9z4b19z9e6eze6ece92372f6
Lee, E. J., Choi, H., Min, K. H., Park, H. W., & Pack, S. P. (2026) · International Journal of Molecular Sciences · doi:10.3390/ijms27083610
Lox, M., & Parac‐Vogt, T. N. (2026) · Journal of the American Chemical Society · doi:10.1021/jacs.6c08412
Augmenting Middle‐Down Sequence Coverage With Trapped Ion Mobility Spectrometry
Reddy, R. S., & Borotto, N. B. (2026) · Journal of Mass Spectrometry · doi:10.1002/jms.70127
Metadata
- Authors
- Cristobal, A., Marino, F., Post, H., van den Toorn, H. W. P., Mohammed, S., & Heck, A. J. R.
- Affiliations
- Utrecht University (NL)
- Utrecht University , , Padualaan 8 , ,
- Corresponding author
- Mohammed, Shabaz
- Heck, Albert J. R.
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2017-02-24
- Volume
- 89
- Issue
- 6
- Pages
- 3318-3325
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC5362747
- OpenAlex
- W2590064181
- Cited by
- 130
- References
- 62
- Citations by year
- 2026: 7 · 2025: 17 · 2024: 8 · 2023: 20 · 2022: 8 · 2021: 17 · 2020: 18 · 2019: 17 · 2018: 11 · 2017: 5
- FWCI
- 6.88
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Locations
- Analytical Chemistry (publishedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Utrecht University Repository (Utrecht University) (submittedVersion, other-oa)
- Funders
- Horizon 2020 Framework Programme (686547)
- Nederlandse Organisatie voor Wetenschappelijk Onderzoek (184.032.201, 700.10.429, 718.015.003)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
- Keywords
- middle-down proteomics, reversed-phase liquid chromatography, electron transfer dissociation, post-translational modification, sequencing depth
- MeSH terms
- HeLa Cells, Humans, Particle Size, Peptide Hydrolases, Peptides, Mass Spectrometry, Proteomics
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