Skip to main content
SpringerLink
Log in

Selected Bioinformatic Tools and MS (MALDI-TOF, PMF) Techniques Used in the Strategy for the Identification of Oat Proteins After 2-DE

  • Protocol
  • First Online:
Oat

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1536))

Abstract

Computer analysis of protein maps obtained from the separation of proteins with two-dimensional polyacrylamide gel electrophoresis (2-DE), in combination with mass spectrometry (MS) analysis and selected bioinformatic tools is used in the strategy for the identification of oat proteins. In proteomic research the most often used MS technique is the combination of ion sources: matrix-assisted laser desorption/ionization (MALDI) and the analyzer of the time of flight (TOF), i.e., MALDI-TOF MS.

This chapter describes the possibilities of the use of selected bioinformatic tools (UniProtKB database, ProtParam, Compute pI/MW programs) for initial identification of separated oat proteins (especially prolamin fractions) with the 2-DE technique. Also the procedure of preparation of samples obtained from cut out protein spots for analysis with the MALDI-TOF MS and peptide mass fingerprinting (PMF) technique is presented.

Among oat prolamins separated with the 2-DE technique (see Chapter 17), 13 protein spots are considered to be the most characteristic (range of MW 27.0–34.6 kDa, pI 5.7–7.6) for this fraction of proteins. Among them there are four protein spots (MW 27.0–28.0 kDa) and two spots (MW 31.4–32.1 kDa) which can correspond to avenins (Accession numbers (AC) in UniProtKB: L0L5I0, I4EP88, I4EP64, L0L4I8 and F2Q9W5, L0L6J0, respectively).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 119.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 159.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. The Universal Protein Resource (UniProt) (2002). http://www.uniprot.org. Accessed Nov 2015

  2. The ProtParam program. http://web.expasy.org/cgi-bin/protparam/protparam. Accessed Nov 2015

  3. The ProtParam program. http://web.expasy.org/protparam/. Accessed Nov 2015

  4. The ProtParam. Documentation is available at http://web.expasy.org/protparam/protparam-doc.html. Accessed Nov 2015

  5. The Compute pI/MW program. http://web.expasy.org/cgi-bin/compute_pi/pi_tool. Accessed Nov 2015

  6. The Compute pI/MW program. http://web.expasy.org/compute_pi/. Accessed Nov 2015

  7. The Compute pI/MW. Documentation is available at http://web.expasy.org/compute_pi/pi_tool-doc.html. Accessed Nov 2015

  8. The UniProt Knowledgebase user manual. http://web.expasy.org/docs/userman.html. Accessed 20 Nov 2015

  9. Pundir S, Magrane M, Martin MJ et al (2015) Searching and navigating UniProt databases. Curr Protoc Bioinform 50:1.27.1–1.27.10. doi:10.1002/0471250953.bi0127s50

    Article  Google Scholar 

  10. Magrane M, UniProt Consortium (2011) UniProt Knowledgebase: a hub of integrated protein data. Database 2011:bar009. doi:10.1093/database/bar009

    Article  PubMed  PubMed Central  Google Scholar 

  11. Jain E, Bairoch A, Duvaud S et al (2009) Infrastructure for the life sciences: design and implementation of the UniProt website. BMC Bioinformatics 10:136. doi:10.1186/1471-2105-10-136

    Article  PubMed  PubMed Central  Google Scholar 

  12. The UniProt Consortium (2008) The Universal Protein Resource (UniProt). Nucleic Acids Res 36(Database issue):D190–D195. doi:10.1093/nar/gkm895

    Google Scholar 

  13. Boutet E, Lieberherr D, Tognolli M et al (2007) UniProtKB/Swiss-Prot. The manually annotated section of the UniProt Knowledgebase. In: Edwards D (ed) Methods in molecular biology, plant bioinformatics: methods and protocols. Humana Press Inc, Totowa, NJ, pp 89–112

    Chapter  Google Scholar 

  14. Boeckmann B, Blatter MC, Famiglietti L et al (2005) Protein variety and functional diversity: Swiss-Prot annotation in its biological context. C R Biol 328(10–11):882–899. doi:10.106/j.crvi.2005.06.001

    Google Scholar 

  15. Apweiler R, Bairoch A, Wu CH (2004) Protein sequence databases. Curr Opin Chem Biol 8(1):76–80. doi:10.1016/j.cbpa.2003.12.004

    Article  CAS  PubMed  Google Scholar 

  16. Apweiler R, Bairoch A, Wu CH et al (2004) UniProt: the Universal Protein knowledgebase. Nucleic Acids Res 32(Database issue):D115–D119. doi:10.1093/nar/gkh131

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Bairoch A, Boeckmann B, Ferro S et al (2004) Swiss-Prot: juggling between evolution and stability. Brief Bioinform 5(1):39–55. doi:10.1093/bib/5.1.39

    Article  CAS  PubMed  Google Scholar 

  18. The Swiss Institute of Bioinformatics (SIB) ExPASy (Expert Protein Analysis System). Bioinformatics Resource Portal. http://www.expasy.org. Accessed Nov 2015

  19. Amersham Biosciences (2003) Ettan MALDI-TOF Pro version 2.0 user manual. Uppsala, Sweden

    Google Scholar 

  20. Amersham Biosciences (2003) Ettan MALDI-TOF method handbook. Uppsala, Sweden

    Google Scholar 

  21. Tanaka K (2003) The origin of macromolecule ionization by laser irradiation (Nobel lecture). Angew Chem Int Ed 42(33):3860–3870. doi:10.1002/anie.200300585

    Article  Google Scholar 

  22. Forner F, Foster LJ, Toppo S (2007) Mass spectrometry data analysis in the proteomics era. Curr Bioinformatics 2(1):63–93. doi:10.2174/157489307779314285

    Article  CAS  Google Scholar 

  23. Trauger SA, Webb W, Siuzdak G (2002) Peptide and protein analysis with mass spectrometry. Spectroscopy 16(1):15–28. doi:10.1155/2002/320152

    Article  CAS  Google Scholar 

  24. Hickman DR, Roepstorff P, Shewry PR et al (1995) Molecular weights of high molecular weight (HMW) subunits of glutenin determined by mass spectrometry. J Cereal Sci 22(2):99–103. doi:10.1016/0733-5210(95)90038-1

    Article  CAS  Google Scholar 

  25. Lim H, Eng J, Yates JR et al (2003) Identification of 2D-gel proteins: a comparison of MALDI/TOF peptide mass mapping to mu LC-ESI tandem mass spectrometry. J Am Soc Mass Spectrom 14(9):957–970. doi:10.1016/s1044-0305(03)00144-2

    Article  CAS  PubMed  Google Scholar 

  26. Salzano AM, Crescenzi M (2005) Mass spectrometry for protein identification and the study of post translational modifications. Ann Ist Super Sanita 41(4):443–450

    PubMed  Google Scholar 

  27. Camafeita E, Solís J, Alfonso P et al (1998) Selective identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of different types of gluten in foods made with cereal mixtures. J Chromatogr A 823(1–2):299–306. doi:10.1016/s0021-9673(98)00621-9

    Article  CAS  PubMed  Google Scholar 

  28. Henzel WJ, Watanabe C, Stults JT (2003) Protein identification: the origins of peptide mass fingerprinting. J Am Soc Mass Spectrom 14(9):931–942. doi:10.1016/s1044-0305(03)00214-9

    Article  CAS  PubMed  Google Scholar 

  29. Thiede B, Höhenwarter W, Krah A et al (2005) Peptide mass fingerprinting. Methods 35(3):237–247. doi:10.1016/j.ymeth.2004.08.015

    Article  CAS  PubMed  Google Scholar 

  30. Perkins DN, Pappin DJ, Creasy DM et al (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20(18):3551–3567. doi:10.1002/(sici)1522-2683(19991201)20:18<3551::aid-elps3551>3.0.c.o;2-2

    Article  CAS  PubMed  Google Scholar 

  31. Matrix Science. http://www.matrixscience.com/search_form_select.html

  32. Galvani M, Hamdan MAA, Righetti PG (2001) Two-dimensional gel electrophoresis/matrix-assisted laser desorption/ionization mass spectrometry of commercial bovine milk. Rapid Commun Mass Spectrom 15(4):258–264. doi:10.1002/rcm.220

    Article  CAS  PubMed  Google Scholar 

  33. Lopez-Ferrer D, Canas B, Vázquez J et al (2006) Sample treatment for protein identification by mass spectrometry-based techniques. Trends Anal Chem 25(10):996–1005. doi:10.1016/j.trac.2006.05.015

    Article  CAS  Google Scholar 

  34. Promega in-gel protein digestion protocol. Trypsin gold, mass spectrometry grade. Technical bulletin available at http://www.promega.com/~/media/Files/Resources/Protocols. Accessed Nov 2015

  35. National Center for Biotechnology Information (NCBI) database. http://www.ncbi.nlm.nih.gov/protein

  36. Gasteiger E, Hoogland C, Gattiker A et al (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics protocols handbook. Humana Press, Totowa, NJ, pp 571–607. doi:10.1385/1-59259-890-0:571

    Chapter  Google Scholar 

  37. The PeptideMass program. http://web.expasy.org/cgi-bin/peptide_mass/peptide-mass.pl. Accessed Nov 2015

  38. The PeptideMass program. http://web.expasy.org/peptide_mass/. Accessed Nov 2015

  39. The PeptideMass. Instructions for PeptideMass peptide characterization software. http://web.expasy.org/peptide_mass/peptide-mass-doc.html. Accessed Nov 2015

  40. Levander F, Rögnvaldsson T, Samuelsson J et al (2004) Automated methods for improved protein identification by peptide mass fingerprinting. Proteomics 4(9):2594–2601. doi:10.1002/pmic.200300804

    Article  CAS  PubMed  Google Scholar 

  41. Bjellqvist B, Hughes GJ, Pasquali C et al (1993) The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis 14(1):1023–1031. doi:10.1002/elps.11501401163

    Article  CAS  PubMed  Google Scholar 

  42. Bjellqvist B, Basse B, Olsen E et al (1994) Reference points for comparisons of two-dimensional maps of proteins from different human cell types defined in a pH scale where isoelectric points correlate with polypeptide compositions. Electrophoresis 15(1):529–539. doi:10.1002/elps.1150150171

    Article  CAS  PubMed  Google Scholar 

  43. Schevchenko A, Wilm M, Vorm O et al (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68(5):850–858. doi:10.1021/ac950914h

    Article  Google Scholar 

  44. Scheler C, Lamer S, Pan Z et al (1998) Peptide mass fingerprint sequence coverage from differently stained proteins on two-dimensional electrophoresis patterns by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Electrophoresis 19(6):918–927. doi:10.1002/elps.1150190607

    Article  CAS  PubMed  Google Scholar 

  45. Canelle L, Pionneau A, Marie A et al (2004) Automating proteome analysis: improvements in throughput, quality and accuracy of protein identification by peptide mass fingerprinting. Rapid Commun Mass Spectrom 18(23):2785–2794. doi:10.1002/rcm.1693

    Article  CAS  PubMed  Google Scholar 

  46. Katayama H, Nagasu T, Oda Y (2001) Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 15(16):1416–1421. doi:10.1002/rcm.379

    Article  CAS  PubMed  Google Scholar 

  47. Song Z, Chen L, Ganapathy A et al (2007) Development and assessment of scoring functions for protein identification using PMF data. Electrophoresis 28(5):864–870. doi:10.1002/elps.200600305

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgment

This work was supported by Ministry of Science and Higher Education, Poland within project No NN 312 286366.

Author information

Authors and Affiliations

  1. Chair of Food Biochemistry, University of Warmia and Mazury in Olsztyn, Plac Cieszyński 1, 10-726, Olsztyn, Poland

    Iwona Szerszunowicz, Dorota Nałęcz & Marta Dziuba

Authors
  1. Iwona Szerszunowicz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dorota Nałęcz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marta Dziuba
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Iwona Szerszunowicz .

Editor information

Editors and Affiliations

  1. Department of Functional Genomics, Plant Breeding & Acclimatization Inst. National Research Institute, Blonie, Poland

    Sebastian Gasparis

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Szerszunowicz, I., Nałęcz, D., Dziuba, M. (2017). Selected Bioinformatic Tools and MS (MALDI-TOF, PMF) Techniques Used in the Strategy for the Identification of Oat Proteins After 2-DE. In: Gasparis, S. (eds) Oat. Methods in Molecular Biology, vol 1536. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6682-0_18

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6682-0_18

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6680-6

  • Online ISBN: 978-1-4939-6682-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation