The DOI matches its Crossref record; 27 of the 27 references it cites were found in a registry or an index.
Detection of Proteome Diversity Resulted from Alternative Splicing is Limited by Trypsin Cleavage Specificity
Open accessAbstract
Alternative splicing dramatically increases transcriptome complexity but its contribution to proteome diversity remains controversial. Exon-exon junction spanning peptides provide direct evidence for the translation of specific splice isoforms and are critical for delineating protein isoform complexity. Here we found that junction-spanning peptides are underrepresented in publicly available mass spectrometry-based shotgun proteomics data sets. Further analysis showed that evolutionarily conserved preferential nucleotide usage at exon boundaries increases the occurrence of lysine- and arginine-coding triplets at the end of exons. Because both lysine and arginine residues are cleavage sites of trypsin, the nearly exclusive use of trypsin as the protein digestion enzyme in shotgun proteomic analyses hinders the detection of junction-spanning peptides. To study the impact of enzyme selection on splice junction detectability, we performed in-silico digestion of the human proteome using six proteases. The six enzymes created a total of 161,125 detectable junctions, and only 1,029 were common across all enzyme digestions. Chymotrypsin digestion provided the largest number of detectable junctions. Our experimental results further showed that combination of a chymotrypsin-based human proteome analysis with a trypsin-based analysis increased detection of junction-spanning peptides by 37% over the trypsin-only analysis and identified over a thousand junctions that were undetectable in fully tryptic digests. Our study demonstrates that detection of proteome diversity resulted from alternative splicing is limited by trypsin cleavage specificity, and that complementary digestion schemes will be essential to comprehensively analyze the translation of alternative splicing isoforms.
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“despite the fact that the number of peptides containing exon-exon splice junctions are reduced with trypsin digestion due to the overabundance of basic amino acid residues at splice junctions [51].”
From Deep proteomic network analysis of Alzheimer’s disease brain reveals alterations in RNA binding proteins and RNA splicing associated with disease (2018) · cites this paper as [51]
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Alternative isoform regulation in human tissue transcriptomes
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A cross-platform toolkit for mass spectrometry and proteomics
Chambers, M., MacLean, B., Burke, R., Amodei, D., Ruderman, D., Neumann, S., Gatto, L., Fischer, B., Pratt, B. S., Egertson, J. D., et al. (2012) · Nature Biotechnology · doi:10.1038/nbt.2377 · Cited by 4,189
Self-citationMass-spectrometry-based draft of the human proteome
Wilhelm, M., Schlegl, J., Hahne, H., Gholami, A. M., Lieberenz, M., Savitski, M. M., Ziegler, E., Butzmann, L., Gessulat, S., Marx, H., et al. (2014) · Nature · doi:10.1038/nature13319 · Cited by 1,820
Proteogenomic characterization of human colon and rectal cancer
CPTAC, T. N., Zhang, B., Wang, J., Wang, X., Zhu, J., Liu, Q., Shi, Z., Chambers, M. C., Zimmerman, L. J., Shaddox, K. F., et al. (2014) · Nature · doi:10.1038/nature13438 · Cited by 1,462
Self-citationTrypsin Cleaves Exclusively C-terminal to Arginine and Lysine Residues
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MS-GF+ makes progress towards a universal database search tool for proteomics
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Self-citationHow did alternative splicing evolve?
Ast, G. (2004) · Nature Reviews Genetics · doi:10.1038/nrg1451 · Cited by 544
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Alternative Splicing May Not Be the Key to Proteome Complexity
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Ran, X., Hu, F., Mao, N., Ruan, Y., Yi, F., Niu, X., Huang, S., Li, S., You, L., Zhang, F., et al. (2021) · Figshare · doi:10.6084/m9.figshare.16557257
Additional file 4 of The evolution of alternative splicing in glioblastoma under therapy
Wang, L., Shamardani, K., Babikir, H. A., Catalan, F. L., Nejo, T., Chang, S., Phillips, J. J., Okada, H., & Diaz, A. A. (2021) · Figshare · doi:10.6084/m9.figshare.13647131
In silico and in cellulo approaches for functional annotation of human protein splice variants
Kiseleva, O. I., Arzumanian, V. A., Kurbatov, I. Y., & Poverennaya, E. V. (2024) · Biomeditsinskaya Khimiya · doi:10.18097/pbmc20247005315
Additional file 2 of The evolution of alternative splicing in glioblastoma under therapy
Wang, L., Shamardani, K., Babikir, H. A., Catalan, F. L., Nejo, T., Chang, S., Phillips, J. J., Okada, H., & Diaz, A. A. (2021) · Figshare · doi:10.6084/m9.figshare.13647125
Additional file 1 of Expression and splicing mediate distinct biological signals
Dam, S. H., Olsen, L. R., & Vitting-Seerup, K. (2023) · Figshare · doi:10.6084/m9.figshare.24406762
Additional file 10 of The evolution of alternative splicing in glioblastoma under therapy
Wang, L., Shamardani, K., Babikir, H. A., Catalan, F. L., Nejo, T., Chang, S., Phillips, J. J., Okada, H., & Diaz, A. A. (2021) · Figshare · doi:10.6084/m9.figshare.13647116
Additional file 2 of Expression and splicing mediate distinct biological signals
Dam, S. H., Olsen, L. R., & Vitting-Seerup, K. (2024) · Figshare · doi:10.6084/m9.figshare.24406944
Ran, X., Hu, F., Mao, N., Ruan, Y., Yi, F., Niu, X., Huang, S., Li, S., You, L., Zhang, F., et al. (2021) · Figshare · doi:10.6084/m9.figshare.16557266
The extended Split-ORF pipeline: Prediction and evaluation of Split-ORFs using Ribo-seq dataPreprint
Kalk, C., Murtagh, J., Despic, V., Müller-McNicoll, M., & Schulz, M. H. (2026) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2026.05.22.727176
“Furthermore, Wang et al. (47) demonstrated that the tryptic cleavage used for most proteomics datasets limits the detection of alternatively spliced isoforms due to evolutionary conserved preference of lysin and arginine codons at exon boundaries.”
IsoPepTracker: An interactive web application for peptide-driven isoform analysis
Mahmud, A., & (202847), C. H. (2026) · PLoS Computational Biology · doi:10.1371/journal.pcbi.1014324
“The authors have declared that no competing interests exist. of junction-spanning peptides [26].”
Cross‐Level Regulatory Interactions Underlying Human Immune Aging
Wu, Q., Zhang, B., Zhi, X., Zhang, Q., Zhang, K., Wang, Y., Zhang, K., Feng, L., Cheng, S., & Xiao, T. (2026) · MedComm · doi:10.1002/mco2.70817
“However, only 224 genes had more than one quantified isoform, consistent with the well-known limitation of shotgun proteomics that many transcript isoforms do not generate isoform-unique tryptic peptides (or generate peptides that are too short or of too low abundance to be detected) [ 25, 26 ].”
Self-citationCommunity-driven advances in computational mass spectrometry: The perspective of EuBIC-MS members
Winkelhardt, D., Schessner, J. P., Berres, S., Nameni, A., Soleymaniniya, A., Schröder, A., Jachmann, C., Schulte, D., Wallmann, G., Webel, H. E., et al. (2026) · Journal of Proteomics · doi:10.1016/j.jprot.2026.105708
Single residues within the YTHDF intrinsically disordered region control m 6 A-dependent mRNA decayPreprint
Ricci, B., Sommer, D., Narvaez, D., Zhang, R., Geri, J. B., & Zaccara, S. (2026) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2026.09.18.752746 · Cited by 1
“However, neither site has been annotated as phosphorylated in available proteomic datasets 37 , largely because the surrounding IDR sequence lacks nearby tryptic cleavage sites, which may limit the detection of peptides in conventional trypsin-based phosphoproteomics 68 .”
Metadata
- Authors
- Wang, X., Codreanu, S. G., Wen, B., Li, K., Chambers, M. C., Liebler, D. C., & Zhang, B.
- Affiliations
- Baylor College of Medicine (US)
- Vanderbilt University (US)
- BGI Group (China) (CN)
- Corresponding author
- Zhang, Bing
- ORCID
- Venue
- Molecular & Cellular Proteomics
- Publisher
- Elsevier BV
- Published
- 2018-03
- Volume
- 17
- Issue
- 3
- Pages
- 422-430
- ISSN
- 1535-9476 (ISSN-L)
- PMC
- PMC5836368
- OpenAlex
- W2773925504
- Cited by
- 97
- References
- 27
- Citations by year
- 2026: 10 · 2025: 6 · 2024: 11 · 2023: 13 · 2022: 13 · 2021: 11 · 2020: 11 · 2019: 11 · 2018: 10
- FWCI
- 4.45
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Molecular & Cellular Proteomics (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- HHS NIH National Cancer Institute (NCI) (U24CA159988, U24CA210954)
- Cancer Prevention and Research Institute of Texas (CPRIT) (RR160027)
- 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 › RNA Research and Splicing
- Keywords
- alternative splicing, proteome diversity, protease selection, chymotrypsin digestion, protein isoforms, in silico digestion, human proteome
- MeSH terms
- Exons, Humans, Neoplasm Proteins, Neoplasms, Peptide Hydrolases, Peptides, Alternative Splicing, Proteome, Cell Line, Tumor
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