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The Current State-of-the-Art Identification of Unknown Proteins Using Mass Spectrometry Exemplified on De Novo Sequencing of a Venom Protease from Bothrops moojeni

(1) Background: The amino acid sequence elucidation of peptides from the gas phase fragmentation mass spectra, de novo sequencing, is a valuable method for the identification of unknown proteins complementary to Edman sequencing. It is increasingly used in shot-gun mass spectrometry (MS)-based prote...

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Autores principales: König, Simone, Obermann, Wolfgang M. J., Eble, Johannes A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9370501/
https://www.ncbi.nlm.nih.gov/pubmed/35956926
http://dx.doi.org/10.3390/molecules27154976
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author König, Simone
Obermann, Wolfgang M. J.
Eble, Johannes A.
author_facet König, Simone
Obermann, Wolfgang M. J.
Eble, Johannes A.
author_sort König, Simone
collection PubMed
description (1) Background: The amino acid sequence elucidation of peptides from the gas phase fragmentation mass spectra, de novo sequencing, is a valuable method for the identification of unknown proteins complementary to Edman sequencing. It is increasingly used in shot-gun mass spectrometry (MS)-based proteomics experiments. We review the current state-of-the-art and use the identification of an unknown snake venom protein targeting the human tissue factor (TF) as an example to describe the analysis process based on manual spectrum interrogation. (2) Methods: The immobilized TF was incubated with a crude B. moojeni venom solution. The potential binding partners were eluted and further purified by gel electrophoresis. Edman degradation was performed to elucidate the N-terminus of the 31 kDa protein of interest. High-resolution MS with collision-induced dissociation was employed to generate peptide fragmentation spectra. Sequence tags were deduced and used for searches in the NCBI and Uniprot databases. Protein matches from the snake species were further validated by target MS/MS. (3) Results: Sequence tag D [K/Q] D [I/L] VDD [K/Q] led to a snake venom serine protease (SVSP) from lancehead B. jararaca (P81824). With target MS/MS, 24% of the SVSP sequence were confirmed; an additional 41% were tentatively assigned by data-independent MS. Edman sequencing provided information for 10 N-terminal amino acid residues, also confirming the match to SVSP. (4) Conclusions: The identification of unknown proteins continues to be a challenge despite major advances in MS instrumentation and bioinformatic tools. The main requirement is the generation of meaningful, high-quality MS peptide fragmentation spectra. These are used to elucidate sufficiently long sequence tags, which can subsequently be submitted to searches in protein databases. This basic method does not require extensive bioinformatics because peptide MS/MS spectra, especially of doubly-charged ions, can be analysed manually. We demonstrated the procedure with the elucidation of SVSP. While de novo sequencing quickly indicates the correct protein group, the validation of the entire protein sequence of amino acid-by-amino acid will take time. Reasons are the need to properly assign isobaric amino acid residues and modifications. With the ongoing efforts in genomics and transcriptomics and the availability of ever more data in public databases, the need for de novo MS sequencing will decrease. Still, not every animal and plant species will be sequenced, so the combination of MS and Edman sequencing will continue to be of importance for the identification of unknown proteins.
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spelling pubmed-93705012022-08-12 The Current State-of-the-Art Identification of Unknown Proteins Using Mass Spectrometry Exemplified on De Novo Sequencing of a Venom Protease from Bothrops moojeni König, Simone Obermann, Wolfgang M. J. Eble, Johannes A. Molecules Review (1) Background: The amino acid sequence elucidation of peptides from the gas phase fragmentation mass spectra, de novo sequencing, is a valuable method for the identification of unknown proteins complementary to Edman sequencing. It is increasingly used in shot-gun mass spectrometry (MS)-based proteomics experiments. We review the current state-of-the-art and use the identification of an unknown snake venom protein targeting the human tissue factor (TF) as an example to describe the analysis process based on manual spectrum interrogation. (2) Methods: The immobilized TF was incubated with a crude B. moojeni venom solution. The potential binding partners were eluted and further purified by gel electrophoresis. Edman degradation was performed to elucidate the N-terminus of the 31 kDa protein of interest. High-resolution MS with collision-induced dissociation was employed to generate peptide fragmentation spectra. Sequence tags were deduced and used for searches in the NCBI and Uniprot databases. Protein matches from the snake species were further validated by target MS/MS. (3) Results: Sequence tag D [K/Q] D [I/L] VDD [K/Q] led to a snake venom serine protease (SVSP) from lancehead B. jararaca (P81824). With target MS/MS, 24% of the SVSP sequence were confirmed; an additional 41% were tentatively assigned by data-independent MS. Edman sequencing provided information for 10 N-terminal amino acid residues, also confirming the match to SVSP. (4) Conclusions: The identification of unknown proteins continues to be a challenge despite major advances in MS instrumentation and bioinformatic tools. The main requirement is the generation of meaningful, high-quality MS peptide fragmentation spectra. These are used to elucidate sufficiently long sequence tags, which can subsequently be submitted to searches in protein databases. This basic method does not require extensive bioinformatics because peptide MS/MS spectra, especially of doubly-charged ions, can be analysed manually. We demonstrated the procedure with the elucidation of SVSP. While de novo sequencing quickly indicates the correct protein group, the validation of the entire protein sequence of amino acid-by-amino acid will take time. Reasons are the need to properly assign isobaric amino acid residues and modifications. With the ongoing efforts in genomics and transcriptomics and the availability of ever more data in public databases, the need for de novo MS sequencing will decrease. Still, not every animal and plant species will be sequenced, so the combination of MS and Edman sequencing will continue to be of importance for the identification of unknown proteins. MDPI 2022-08-05 /pmc/articles/PMC9370501/ /pubmed/35956926 http://dx.doi.org/10.3390/molecules27154976 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Review
König, Simone
Obermann, Wolfgang M. J.
Eble, Johannes A.
The Current State-of-the-Art Identification of Unknown Proteins Using Mass Spectrometry Exemplified on De Novo Sequencing of a Venom Protease from Bothrops moojeni
title The Current State-of-the-Art Identification of Unknown Proteins Using Mass Spectrometry Exemplified on De Novo Sequencing of a Venom Protease from Bothrops moojeni
title_full The Current State-of-the-Art Identification of Unknown Proteins Using Mass Spectrometry Exemplified on De Novo Sequencing of a Venom Protease from Bothrops moojeni
title_fullStr The Current State-of-the-Art Identification of Unknown Proteins Using Mass Spectrometry Exemplified on De Novo Sequencing of a Venom Protease from Bothrops moojeni
title_full_unstemmed The Current State-of-the-Art Identification of Unknown Proteins Using Mass Spectrometry Exemplified on De Novo Sequencing of a Venom Protease from Bothrops moojeni
title_short The Current State-of-the-Art Identification of Unknown Proteins Using Mass Spectrometry Exemplified on De Novo Sequencing of a Venom Protease from Bothrops moojeni
title_sort current state-of-the-art identification of unknown proteins using mass spectrometry exemplified on de novo sequencing of a venom protease from bothrops moojeni
topic Review
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9370501/
https://www.ncbi.nlm.nih.gov/pubmed/35956926
http://dx.doi.org/10.3390/molecules27154976
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