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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The number of peptides containing exon-exon splice junctions is reduced with trypsin digestion due to the overabundance of basic amino acid residues at splice junctions.
“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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“Therefore, sampling peptides from the small space of unique amino acids that can directly confirm the presence of a protein isoform is limited by the space of “informa-tive” (i.e. unique-to-an-isoform) peptides[55].”
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Assessing conservation of alternative splicing with evolutionary splicing graphsPreprint
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“According to Wang et al. [46], the proteases most used in in vitro studies for the simulation of gastrointestinal digestion are pepsin, pancreatin (which is a mixture of several digestive enzymes, including trypsin and chymotrypsin), and chymotrypsin.”
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Tools and tactics for studying alternative splicing
Sousa‐Luís, R., & Carmo‐Fonseca, M. (2026) · Nature Reviews Genetics · doi:10.1038/s41576-026-00952-4 · Cited by 8 · see citing works
Kim, C.-Y., Na, K., Park, S., Jeong, S., Cho, J.-Y., Shin, H.-O., Lee, M. J., Han, G., & Paik, Y. (2019) · Molecular & Cellular Proteomics · doi:10.1074/mcp.ra119.001456 · Cited by 9 · see citing works
“Studies have shown that the percentages of Lys and Arg that constitute the exon boundary are relatively high and that chymotrypsin and Asp-N greatly improves the peptide coverage of the exon-exon splice junction containing K/R residue (66).”
BackgroundMethodBeyond the gene: decoding alternative isoforms
Mattioli, K., & Bulyk, M. L. (2025) · Trends in Genetics · doi:10.1016/j.tig.2025.11.003 · Cited by 9 · see citing works
Impact of alternative splicing on Arabidopsis proteome
Reyes, A. V., Zhang, C., Karunadasa, S. S., Shrestha, R., Grismer, T. S., Byun, D., & Xu, S. (2026) · Nucleic Acids Research · doi:10.1093/nar/gkag400 · Cited by 7 · see citing works
“As noted previously, IR-derived peptides are often challenging to detect because exon–intron junctions are frequently enriched in K/R residues, which can obscure the identification of AS-specific peptides [ 42 , 44 ].”
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APOER2 splicing repertoire in Alzheimer’s disease: Insights from long-read RNA sequencing
Gallo, C. M., Kistler, S. A., Natrakul, A., Labadorf, A. T., Beffert, U., & Ho, A. (2024) · PLoS Genetics · doi:10.1371/journal.pgen.1011348 · Cited by 5 · see citing works
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Validating Amino Acid Variants in Proteogenomics Using Sequence Coverage by Multiple Reads
Levitsky, L. I., Kuznetsova, K. G., Kliuchnikova, A. A., Ilina, I. Y., Goncharov, A. O., Lobas, A. A., Ivanov, M. V., Lazarev, V. N., Ziganshin, R., Gorshkov, M. V., et al. (2022) · Journal of Proteome Research · doi:10.1021/acs.jproteome.2c00033 · Cited by 5 · see citing works
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“Secondly, we observed very little peptide support for our novel isoforms, likely due to trypsin digestion in shotgun proteomics disfavouring the creation of peptides that span exon junctions (Wang et al. 2018).”
A ratiometric catalog of protein isoform shifts in the cardiac fetal gene program
Han, Y., Binti, S., Wennersten, S. A., Pandi, B., Ng, D. C. M., Lau, E., & Lam, M. P. Y. (2025) · JCI Insight · doi:10.1172/jci.insight.184309 · Cited by 5 · see citing works
“At present, the identification of splicing-derived isoform proteins (spliceoforms) remains hindered by experimental and data analytical challenges (42, 59, 60).”
Alternative splicing in plants: emerging order from chaos
Jabre, I., Khokhar, W., Reddy, A. S. N., Wilson, C. M., & Syed, N. H. (2026) · Trends in Plant Science · doi:10.1016/j.tplants.2026.04.001 · Cited by 5 · see citing works
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Self-citationIS-PRM-Based Peptide Targeting Informed by Long-Read Sequencing for Alternative Proteome Detection
Korchak, J. A., Jeffery, E. D., Bandyopadhyay, S., Jordan, B. T., Lehe, M. D., Watts, E. F., Fenix, A. M., Wilhelm, M., & Sheynkman, G. (2024) · Journal of the American Society for Mass Spectrometry · doi:10.1021/jasms.4c00119 · Cited by 5 · see citing works
Pandi, B., Brenman, S., Black, A., Ng, D. C. M., Lau, E., & Lam, M. P. Y. (2024) · Journal of Proteome Research · doi:10.1021/acs.jproteome.3c00789 · Cited by 3 · see citing works
Horokhovskyi, Y., Roetschke, H. P., Cormican, J. A., Pašen, M., Garazhian, S., Mishto, M., & Liepe, J. (2025) · Molecular & Cellular Proteomics · doi:10.1016/j.mcpro.2025.101039 · Cited by 3 · see citing works
“…with proteins that are enzymatically digested into peptides—for example, by trypsin, which mainly cleaves proteins after arginine (R) and lysin (K) (41–43), separated by chromatography, and measured with tandem MS. Peptide sequences are matched to a reference protein sequence database for…”
Detecting Differential Alternative Splicing in Mass Spectrometry-based Proteomics DataPreprint
Ammar, C., Csaba, G., Hadziahmetovic, A., Vasilopoulou, C. G., Gruber, M., Mann, M., & Zimmer, R. (2023) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2023.09.19.558203 · Cited by 3 · see citing works
Decoding the Cryptic Proteome Between Antigens and Novel Functional Proteins
Bawden, E., Amigorena, S., & Arribas, Y. A. (2025) · European Journal of Immunology · doi:10.1002/eji.70102 · Cited by 2 · see citing works
“In addition, trypsin digestion poses limitations on the identification of cryptic peptides derived from splicing junctions because exon boundaries exhibit high usage of arginine and lysine [14].”
Wang, Z.-Y., Zhang, W., Zhang, N., Wu, P.-M., Su, Q., Ma, C., Wang, W., & Chen, M. (2026) · Journal of Experimental Botany · doi:10.1093/jxb/erag052 · Cited by 2 · see citing works
Lehe, M. D., Almofeez, R., Jeffery, E. D., & Sheynkman, G. (2026) · Journal of Mass Spectrometry · doi:10.1002/jms.70024 · Cited by 3 · see citing works
Lobas, A. A., Solovyeva, E. M., Levitsky, L. I., Goncharov, A. O., Lyssuk, E. Y., Larin, S. S., Moshkovskii, S. A., & Gorshkov, M. V. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms24032466 · Cited by 2 · see citing works
“There are a number of reasons for such dichotomy in mass spectrometry-based bottom-up proteomics, including (i) low protein sequence coverage translating into a low probability to detect splicing-specific peptides, (ii) a statistically increased probability of arginine or lysine being encoded at exon boundaries [37],…”
BackgroundNarykov, O., Johnson, N. T., & Korkin, D. (2018) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/256834 · Cited by 2 · see citing works
“and carrying out other basic functions (2-4).”
MethodZhang, F., Tang, L., Ran, X., Mao, N., Ruan, Y., Yi, F., Liu, C., Niu, X., Huang, S., Li, S., et al. (2019) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/547810 · Cited by 2 · see citing works
Expression and Splicing Mediate Distinct Biological SignalsPreprint
Dam, S. H., Olsen, L. R., & Vitting‐Seerup, K. (2022) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2022.08.29.505720 · Cited by 2 · see citing works
Zhao, F., Yan, J., Liu, Z., Pan, D., Wang, Y., Li, L., Suo, S., Dang, K., Li, D., Dang, Y., et al. (2025) · Food Research International · doi:10.1016/j.foodres.2025.118157 · Cited by 1 · see citing works
Assessing Conservation of Alternative Splicing with Evolutionary Splicing GraphsPreprint
Zea, D. J., Laskina, S., Baudin, A., Richard, H., & Laine, É. (2020) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2020.11.14.382820 · Cited by 1 · see citing works
Zhong, J. J., Morin, G. B., & Chen, D. D. Y. (2022) · Wiley eBooks · doi:10.1002/9783527833092.ch3 · Cited by 1 · see citing works
Gallo, C. M., Kistler, S. A., Natrakul, A., Labadorf, A. T., Beffert, U., & Ho, A. (2023) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2023.01.12.523804 · Cited by 1 · see citing works
“Although detection of lowly expressed isoforms at the protein level can be technically challenging (Blakeley et al., 2010; X. Wang et al., 2018), novel methods including the integration of RNA-seq and mass spectrometry data (Agosto et al., 2019; Han et al., 2020) are rapidly increasing the…”
MethodAit-hamlat, A., Zea, D. J., Labeeuw, A., Polit, L., Richard, H., & Laine, É. (2017) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/119891
Proteomics and Metabolomics in Pain
Schmidt, M. (2020) · Elsevier eBooks · doi:10.1016/b978-0-12-809324-5.24256-9
Data regarding the sensibility to proteolysis of a natural apolipoprotein A-I mutant.
Gaddi, G. M., Gisonno, R. A., Rosu, S. A., Cortez, M. F., Finarelli, G. S., Ramella, N. A., & Tricerri, M. A. (2020) · Data in Brief · doi:10.1016/j.dib.2020.105960
“Together with trypsin, this enzyme is a potent tool for structural studies involving mass spectrometry and cross-linking [2] , as the cleavage pattern is well-defined (it selectively hydrolyzes peptide bonds on the C-terminal side of tyrosine, phenylalanine, tryptophan, and leucine) [3] .”
BackgroundSegelcke, D., van der Burgt, M., Kappert, C., Garcia, D. S., Sondermann, J. R., Bigalke, S., Pradier, B., Gómez‐Varela, D., Zahn, P. K., Schmidt, M., et al. (2022) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2022.03.07.482980
Additional file 1 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.13647119
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.16557251
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.16557254
Additional file 7 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.13647140
Additional file 6 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.13647137
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.16557245
Additional file 8 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.13647143
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.16557263
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.16557248
Additional file 3 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.13647128
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.16557260
Additional file 5 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.13647134
Additional file 9 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.13647146
- 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 · 2017: 1
- 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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