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Cited by 5Advanced Proteomics Techniques and ApplicationsMass Spectrometry Techniques and ApplicationsVenomous Animal Envenomation and Studies

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SEQUENCE SLIDER: integration of structural and genetic data to characterize isoforms from natural sources

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Borges, Rafael J, Salvador, Guilherme H M, Pimenta, Daniel C, dos Santos, Lucilene D, Fontes, Marcos R M, Usón, Isabel·2022·Nucleic Acids Research
DOI 10.1093/nar/gkac029PMC PMC9122596

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Abstract

Proteins isolated from natural sources can be composed of a mixture of isoforms with similar physicochemical properties that coexist in the final steps of purification. Yet, even where unverified, the assumed sequence is enforced throughout the structural studies. Herein, we propose a novel perspective to address the usually neglected sequence heterogeneity of natural products by integrating biophysical, genetic and structural data in our program SEQUENCE SLIDER. The aim is to assess the evidence supporting chemical composition in structure determination. Locally, we interrogate the experimental map to establish which side chains are supported by the structural data, and the genetic information relating sequence conservation is integrated into this statistic. Hence, we build a constrained peptide database, containing most probable sequences to interpret mass spectrometry data (MS). In parallel, we perform MS de novo sequencing with genomic-based algorithms to detect point mutations. We calibrated SLIDER with Gallus gallus lysozyme, whose sequence is unequivocally established and numerous natural isoforms are reported. We used SLIDER to characterize a metalloproteinase and a phospholipase A2-like protein from the venom of Bothrops moojeni and a crotoxin from Crotalus durissus collilineatus. This integrated approach offers a more realistic structural descriptor to characterize macromolecules isolated from natural sources.

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Crossref · 10.1093/nar/gkac029

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Figures

Graphical Abstract SEQUENCE SLIDER method integrates structural (blue), mass spectrometry (orange) and phylogenetic (magenta) data of protein sample purified from natural sources to assign isoform amino acid sequence (green).
Graphical Abstract SEQUENCE SLIDER method integrates structural (blue), mass spectrometry (orange) and phylogenetic (magenta) data of protein sample purified from natural sources to assign isoform amino acid sequence (green).CC BY-NC · PubMed Central
Figure 1. Scheme of how SEQUENCE SLIDER integrates structural, mass spectrometry and phylogenetic data for sequence assignment.
Figure 1. Scheme of how SEQUENCE SLIDER integrates structural, mass spectrometry and phylogenetic data for sequence assignment.CC BY-NC · PubMed Central
Figure 2. Sequence assignment of lysozyme. (A) Mass spectrometry data are used against an NCBI and UniProt database of lysozyme sequences, against PEAKS/SPIDER evaluation and against PatternLab with database built from crystallographic data and the sequence from the crystal model. (B) Database constructed by SEQUENCE SLIDER from crystallographic data.
Figure 2. Sequence assignment of lysozyme. (A) Mass spectrometry data are used against an NCBI and UniProt database of lysozyme sequences, against PEAKS/SPIDER evaluation and against PatternLab with database built from crystallographic data and the sequence from the crystal model. (B) Database constructed by SEQUENCE SLIDER from crystallographic data.CC BY-NC · PubMed Central
Figure 3. Electron density of omit map of different lysozyme residues in comparison to the PDB code: 6G8A with mass spectrometry and phylogenetic analysis statistics. RT stands for residue type; RSCC for real-space correlation coefficient, PSc for Primary Score, %PhA frequency from phylogenetic analysis; and ! for SLIDER choice for given amino acid in a single letter code.
Figure 3. Electron density of omit map of different lysozyme residues in comparison to the PDB code: 6G8A with mass spectrometry and phylogenetic analysis statistics. RT stands for residue type; RSCC for real-space correlation coefficient, PSc for Primary Score, %PhA frequency from phylogenetic analysis; and ! for SLIDER choice for given amino acid in a single letter code.CC BY-NC · PubMed Central
Figure 4. Sequence modelling of BaM. Three techniques, mass spectrometry (with PatternLab and PEAKS/SPIDER evaluation), phylogenetic and crystallographic analysis are integrated to assign the BaM sequence.
Figure 4. Sequence modelling of BaM. Three techniques, mass spectrometry (with PatternLab and PEAKS/SPIDER evaluation), phylogenetic and crystallographic analysis are integrated to assign the BaM sequence.CC BY-NC · PubMed Central
Figure 5. Sequence heterogeneity modelling of BmooMP-I. (A) Mass spectrometry data against an NCBI and UniProt database with Bothrops genus sequences, against PEAKS/SPIDER evaluation and against PatternLab with database built from crystallography and the sequence from the crystal model. (B) Database constructed by SEQUENCE SLIDER from crystallographic data. Adapted from (53).
Figure 5. Sequence heterogeneity modelling of BmooMP-I. (A) Mass spectrometry data against an NCBI and UniProt database with Bothrops genus sequences, against PEAKS/SPIDER evaluation and against PatternLab with database built from crystallography and the sequence from the crystal model. (B) Database constructed by SEQUENCE SLIDER from crystallographic data. Adapted from (53).CC BY-NC · PubMed Central
Figure 6. Electron density of omit map with possible residues given crystallographic, mass spectrometry and phylogenetic analysis statistics in metalloproteinase BmooMP-I. RT stands for residue type; RSCC for real-space correlation coefficient, PSc for Primary Score, %PhA frequency from phylogenetic analysis; and ! for SLIDER choice for given amino acid in a single letter code.
Figure 6. Electron density of omit map with possible residues given crystallographic, mass spectrometry and phylogenetic analysis statistics in metalloproteinase BmooMP-I. RT stands for residue type; RSCC for real-space correlation coefficient, PSc for Primary Score, %PhA frequency from phylogenetic analysis; and ! for SLIDER choice for given amino acid in a single letter code.CC BY-NC · PubMed Central
Figure 7. Sequence heterogeneity modelling of MjTX-I. (A) Mass spectrometry data against an NCBI database with Bothrops genus sequences, against PEAKS/SPIDER evaluation and against PatternLab with database built from crystallographic data and the sequence from the crystal model. (B) Database constructed by SEQUENCE SLIDER from crystallographic data.
Figure 7. Sequence heterogeneity modelling of MjTX-I. (A) Mass spectrometry data against an NCBI database with Bothrops genus sequences, against PEAKS/SPIDER evaluation and against PatternLab with database built from crystallographic data and the sequence from the crystal model. (B) Database constructed by SEQUENCE SLIDER from crystallographic data.CC BY-NC · PubMed Central
Figure 8. Electron density of omit map of different MjTX-I residues in respect to its previous crystallographic model (PDB code: 6CE2) with mass spectrometry and phylogenetic analysis statistics. RT stands for residue type; RSCC for real-space correlation coefficient, PSc for Primary Score, %PhA frequency from phylogenetic analysis; and ! for SLIDER choice for given amino acid in a single letter code.
Figure 8. Electron density of omit map of different MjTX-I residues in respect to its previous crystallographic model (PDB code: 6CE2) with mass spectrometry and phylogenetic analysis statistics. RT stands for residue type; RSCC for real-space correlation coefficient, PSc for Primary Score, %PhA frequency from phylogenetic analysis; and ! for SLIDER choice for given amino acid in a single letter code.CC BY-NC · PubMed Central
Figure 9. Sequence heterogeneity modelling of CBCol. (A) Mass spectrometry data are used against an NCBI and UniProt database with Crotalus genus sequences, against PEAKS/SPIDER evaluation and against PatternLab with database built from crystallographic data and the sequence from the crystal model. (B) Database constructed by SEQUENCE SLIDER from crystallographic data.
Figure 9. Sequence heterogeneity modelling of CBCol. (A) Mass spectrometry data are used against an NCBI and UniProt database with Crotalus genus sequences, against PEAKS/SPIDER evaluation and against PatternLab with database built from crystallographic data and the sequence from the crystal model. (B) Database constructed by SEQUENCE SLIDER from crystallographic data.CC BY-NC · PubMed Central
Figure 10. Electron density of omit map of different CbCol residues among its monomers with mass spectrometry and phylogenetic analysis statistics. RSCC stands for real-space correlation coefficient, PSc for Primary Score and %PhA frequency from phylogenetic analysis.
Figure 10. Electron density of omit map of different CbCol residues among its monomers with mass spectrometry and phylogenetic analysis statistics. RSCC stands for real-space correlation coefficient, PSc for Primary Score and %PhA frequency from phylogenetic analysis.CC BY-NC · PubMed Central
Figure 11. Three groups of residues approximately isosteric sharing atomic structure with their molecular mass, coloured in blue and red hydrophilic and hydrophobic residues, respectively.
Figure 11. Three groups of residues approximately isosteric sharing atomic structure with their molecular mass, coloured in blue and red hydrophilic and hydrophobic residues, respectively.CC BY-NC · PubMed Central

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Metadata

Authors
Borges, R. J., Salvador, G. H. M., Pimenta, D. C., dos Santos, L. D., Fontes, M. R. M., & Usón, I.
Affiliations
  • Consejo Superior de Investigaciones Científicas (ES)
  • Departament of Biophysics and Pharmacology, Biosciences Institute, São Paulo State University (UNESP), Botucatu, São Paulo 18618-689, Brazil
  • Instituto Butantan (BR)
  • Universidade Estadual Paulista (Unesp) (BR)
  • Institució Catalana de Recerca i Estudis Avançats (ES)
Corresponding author
Borges, Rafael J
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2022-02-01
Volume
50
Issue
9
Pages
e50-e50
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gkac029
PMC
PMC9122596
OpenAlex
W4210433582
Cited by
5
References
89
Citations by year
2025: 1 · 2024: 2 · 2023: 1 · 2022: 1
FWCI
0.45
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by-nc)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nd)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • LA Referencia (Red Federada de Repositorios Institucionales de Publicaciones Científicas) (publishedVersion, other-oa)
  • DIGITAL.CSIC (Spanish National Research Council (CSIC)) (submittedVersion, cc-by-nc-nd)
Funders
  • São Paulo Research Foundation (2015/17286–0, 2019/05958-4, 2020/10143–7)
  • FAPESP (2016/24191–8)
  • Ministerio de Ciencia e Innovación (PGC2018-101370-BI00 AEI/FEDER/UE)
  • CNPq (301974/2019–5, 302883/2017–7)
Topics
  • Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
  • Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Toxicology › Venomous Animal Envenomation and Studies
Keywords
protein isoforms, sequence heterogeneity, natural products, de novo sequencing, mass spectrometry, sequence conservation, point mutation detection, metalloproteinases, phospholipase A2, crotoxin, Crotalus durissus collilineatus, lysozyme, venom proteins
MeSH terms
Animals, Crotalid Venoms, Crotoxin, Software, Crotalus, Protein Isoforms, Complex Mixtures, Phospholipases A2

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Borges, R. J., Salvador, G. H. M., Pimenta, D. C., dos Santos, L. D., Fontes, M. R. M., & Usón, I. (2022). SEQUENCE SLIDER: integration of structural and genetic data to characterize isoforms from natural sources. Nucleic Acids Research. https://doi.org/10.1093/nar/gkac029

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