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Cited by 113Advanced Proteomics Techniques and ApplicationsMass Spectrometry Techniques and ApplicationsRNA Research and Splicing

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Detection of Proteome Diversity Resulted from Alternative Splicing is Limited by Trypsin Cleavage Specificity

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Wang, Xiaojing, Codreanu, Simona G., Wen, Bo, Li, Kai, Chambers, Matthew C., Liebler, Daniel C., Zhang, Bing·2018·Molecular & Cellular Proteomics
DOI 10.1074/mcp.ra117.000155PMC PMC5836368
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Abstract

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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Figures

Fig. 1. Enrichment of tryptic peptide termini proximal to exon boundaries. Distribution of the relative distance between the locations of the observed peptide termini in the CPTAC_CRC data set and exon boundaries is plotted for (A) all distinct peptides or (B) exon-exon junction peptides. Only peptides that terminate within 10 base pairs of a splicing site were counted. An enrichment of tryptic peptide termini proximal to exon boundaries was observed, most remarkably for the four positions labeled 1–4 in red.
Fig. 1. Enrichment of tryptic peptide termini proximal to exon boundaries. Distribution of the relative distance between the locations of the observed peptide termini in the CPTAC_CRC data set and exon boundaries is plotted for (A) all distinct peptides or (B) exon-exon junction peptides. Only peptides that terminate within 10 base pairs of a splicing site were counted. An enrichment of tryptic peptide termini proximal to exon boundaries was observed, most remarkably for the four positions labeled 1–4 in red.CC BY · PubMed Central
Fig. 2. Enrichment of certain amino acids among exon-ending and exon-exon junction residues. Gray bars depict overall amino acid composition for all human protein sequences, whereas black bars depict amino acid composition for all exon-ending and exon-exon junction residues. Five amino acids indicated by red asterisks were enriched more than 1.5 fold among the exon-ending and junction residues, including glutamine (Q, 2.45 fold), lysine (K, 2.37 fold), glycine (G, 2.26 fold), arginine (R, 1.94 fold), and glutamic acid (E, 1.56 fold).
Fig. 2. Enrichment of certain amino acids among exon-ending and exon-exon junction residues. Gray bars depict overall amino acid composition for all human protein sequences, whereas black bars depict amino acid composition for all exon-ending and exon-exon junction residues. Five amino acids indicated by red asterisks were enriched more than 1.5 fold among the exon-ending and junction residues, including glutamine (Q, 2.45 fold), lysine (K, 2.37 fold), glycine (G, 2.26 fold), arginine (R, 1.94 fold), and glutamic acid (E, 1.56 fold).CC BY · PubMed Central
Fig. 3. Preferential cleavage by trypsin at splice sites. A, Consensus sequences on either side of the exon-intron boundaries for all coding genes in the human genome. The first three rows in the table list the occurrence probabilities of lysine (K) and arginine (R) at splice sites for each of the three reading frames, respectively. As shown in the last row of the table, adjusted for reading frame usage, one quarter of the splicing sites can be recognized by trypsin as potential cleavage sites, which is twice the genome-wide average (11%). B, Two scenarios resulting in the type 1–4 peptides labeled in (a). The vertical dashed lines represent the splicing sites, and the scissors indicate the trypsin-digesting sites. The occurrence probability of scenario 1 is 0.124, in which the digestion produces type 1 and 3 peptides. The occurrence probability of scenario 2 is 0.111, in which type 2 and 4 peptides are generated. C, Consensus sequences on the exon side of five eukaryotic genomes. There is no significant difference between the human and mouse 5′ donor exon. The “AG-R” pattern seems to become more pronounced during evolution.
Fig. 3. Preferential cleavage by trypsin at splice sites. A, Consensus sequences on either side of the exon-intron boundaries for all coding genes in the human genome. The first three rows in the table list the occurrence probabilities of lysine (K) and arginine (R) at splice sites for each of the three reading frames, respectively. As shown in the last row of the table, adjusted for reading frame usage, one quarter of the splicing sites can be recognized by trypsin as potential cleavage sites, which is twice the genome-wide average (11%). B, Two scenarios resulting in the type 1–4 peptides labeled in (a). The vertical dashed lines represent the splicing sites, and the scissors indicate the trypsin-digesting sites. The occurrence probability of scenario 1 is 0.124, in which the digestion produces type 1 and 3 peptides. The occurrence probability of scenario 2 is 0.111, in which type 2 and 4 peptides are generated. C, Consensus sequences on the exon side of five eukaryotic genomes. There is no significant difference between the human and mouse 5′ donor exon. The “AG-R” pattern seems to become more pronounced during evolution.CC BY · PubMed Central
Fig. 4. Venn-diagram showing shared and unique junctions (A) and K/R junctions (B) with peptide evidence in in-silico digestion experiments using trypsin, chymotrypsin and Asp-N. Peptide length was specified to be between 8 and 25 residues.
Fig. 4. Venn-diagram showing shared and unique junctions (A) and K/R junctions (B) with peptide evidence in in-silico digestion experiments using trypsin, chymotrypsin and Asp-N. Peptide length was specified to be between 8 and 25 residues.CC BY · PubMed Central
Fig. 5. Impact of protein digestion enzyme selection on splice junction verification. A, Digestion of RKO protein lysate using trypsin (Try) resulted in a higher proportion of type 1–4 peptides compared with using chymotrypsin (Chy). See Fig. 3B for the graphical representation of the different types of peptides. B, Comparison of the numbers of junctions with supporting peptides identified in trypsin or chymotrypsin digested samples. The left Venn diagram includes all junctions, whereas the right Venn diagram includes only junctions with lysine (K) or arginine (R) at the junction site and which cannot be identified in tryptic digests.
Fig. 5. Impact of protein digestion enzyme selection on splice junction verification. A, Digestion of RKO protein lysate using trypsin (Try) resulted in a higher proportion of type 1–4 peptides compared with using chymotrypsin (Chy). See Fig. 3B for the graphical representation of the different types of peptides. B, Comparison of the numbers of junctions with supporting peptides identified in trypsin or chymotrypsin digested samples. The left Venn diagram includes all junctions, whereas the right Venn diagram includes only junctions with lysine (K) or arginine (R) at the junction site and which cannot be identified in tryptic digests.CC BY · PubMed Central
Fig. 6. Examples showing the effect of trypsin preferential cleavage at junction site on protein isoform identification in the RKO data. Peptides from chymotrypsin digestion are colored in green and those from trypsin are in blue. Boxes in red represent the alternative exons. A, An illustration of exon skipping events in ARFGAP1 gene. B, An illustration of alternative 3′ acceptor site in the NPM1 gene. The isoform 3 utilizes an alternate 3′ terminal exon, resulting in a shorter protein with distinct C terminus compared with isoform 1/2. C, An illustration of an alternative 5′ acceptor site in the ISCU gene. Representative spectra for the 4 green-colored peptide sequences from chymotrypsin digestion are shown on the right.
Fig. 6. Examples showing the effect of trypsin preferential cleavage at junction site on protein isoform identification in the RKO data. Peptides from chymotrypsin digestion are colored in green and those from trypsin are in blue. Boxes in red represent the alternative exons. A, An illustration of exon skipping events in ARFGAP1 gene. B, An illustration of alternative 3′ acceptor site in the NPM1 gene. The isoform 3 utilizes an alternate 3′ terminal exon, resulting in a shorter protein with distinct C terminus compared with isoform 1/2. C, An illustration of an alternative 5′ acceptor site in the ISCU gene. Representative spectra for the 4 green-colored peptide sequences from chymotrypsin digestion are shown on the right.CC BY · PubMed Central

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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)
DOI
10.1074/mcp.ra117.000155
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
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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