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A simple MALDI plate functionalization by Vmh2 hydrophobin for serial multi-enzymatic protein digestions

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Abstract

The development of efficient and rapid methods for the identification with high sequence coverage of proteins is one of the most important goals of proteomic strategies today. The on-plate digestion of proteins is a very attractive approach, due to the possibility of coupling immobilized-enzymatic digestion with direct matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS) analysis. The crucial step in the development of on-plate immobilization is however the functionalization of the solid surface. Fungal self-assembling proteins, the hydrophobins, are able to efficiently functionalize surfaces. We have recently shown that such modified plates are able to absorb either peptides or proteins and are amenable to MALDI-TOF-MS analysis. In this paper, the hydrophobin-coated MALDI sample plates were exploited as a lab-on-plate for noncovalent immobilization of enzymes commonly used in protein identification/characterization, such as trypsin, V8 protease, PNGaseF, and alkaline phosphatase. Rapid and efficient on-plate reactions were performed to achieve high sequence coverage of model proteins, particularly when performing multiple enzyme digestions. The possibility of exploiting this direct on-plate MALDI-TOF/TOF analysis has been investigated on model proteins and, as proof of concept, on entire whey milk proteome.

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References

  1. Angel TE, Aryal UK, Hengel SM et al (2012) Mass spectrometry-based proteomics: existing capabilities and future directions. Chem Soc Rev 41:3912–3928. doi:10.1039/c2cs15331a

    Article  CAS  Google Scholar 

  2. Yamaguchi H, Miyazaki M (2013) Enzyme-immobilized reactors for rapid and efficient sample preparation in MS-based proteomic studies. Proteomics 13:457–466. doi:10.1002/pmic.201200272

    Article  CAS  Google Scholar 

  3. Aebersold R, Mann M (2003) Mass spectrometry-based proteomics. Nature 422:198–207. doi:10.1038/nature01511

    Article  CAS  Google Scholar 

  4. Switzar L, Giera M, Niessen WMA (2013) Protein digestion: an overview of the available techniques and recent developments. J Proteome Res 12:1067–1077. doi:10.1021/pr301201x

    Article  CAS  Google Scholar 

  5. Massolini G, Calleri E (2005) Immobilized trypsin systems coupled on-line to separation methods: recent developments and analytical applications. J Sep Sci 28:7–21. doi:10.1002/jssc.200401941

    Article  CAS  Google Scholar 

  6. Girelli AM, Mattei E (2005) Application of immobilized enzyme reactor in on-line high performance liquid chromatography: a review. J Chromatogr B Analyt Technol Biomed Life Sci 819:3–16. doi:10.1016/j.jchromb.2005.01.031

    Article  CAS  Google Scholar 

  7. Vestling MM, Fenselau C (1994) Poly(vinylidene difluoride) membranes as the interface between laser desorption mass spectrometry, gel electrophoresis, and in situ proteolysis. Anal Chem 66:471–477. doi:10.1021/ac00076a009

    Article  CAS  Google Scholar 

  8. Sun J, Hu K, Liu Y et al (2013) Novel superparamagnetic sanoparticles for trypsin immobilization and the application for efficient proteolysis. J Chromatogr B Analyt Technol Biomed Life Sci 942–943:9–14. doi:10.1016/j.jchromb.2013.10.015

    Article  Google Scholar 

  9. Bao H, Zhang L, Chen G (2013) Immobilization of trypsin via graphene oxide-silica composite for efficient microchip proteolysis. J Chromatogr A 1310:74–81. doi:10.1016/j.chroma.2013.08.040

    Article  CAS  Google Scholar 

  10. Fan C, Shi Z, Pan Y et al (2014) Dual matrix-based immobilized trypsin for complementary proteolytic digestion and fast proteomics analysis with higher protein sequence coverage. Anal Chem 86:1452–1458. doi:10.1021/ac402696b

    Article  CAS  Google Scholar 

  11. Brownridge P, Beynon RJ (2011) The importance of the digest: proteolysis and absolute quantification in proteomics. Methods 54:351–360. doi:10.1016/j.ymeth.2011.05.005

    Article  CAS  Google Scholar 

  12. Liu Y, Lu H, Zhong W et al (2006) Multilayer-assembled microchip for enzyme immobilization as reactor toward low-level protein identification. Anal Chem 78:801–808. doi:10.1021/ac051463w

    Article  CAS  Google Scholar 

  13. Liu Y, Zhong W, Meng S et al (2006) Assembly-controlled biocompatible interface on a microchip: strategy to highly efficient proteolysis. Chemistry 12:6585–6591. doi:10.1002/chem.200501622

    Article  CAS  Google Scholar 

  14. Gao J, Xu J, Locascio LE, Lee CS (2001) Integrated microfluidic system enabling protein digestion, peptide separation, and protein identification. Anal Chem 73:2648–2655

    Article  CAS  Google Scholar 

  15. Qiao L, Liu Y, Hudson SP et al (2008) A nanoporous reactor for efficient proteolysis. Chemistry 14:151–157. doi:10.1002/chem.200701102

    Article  CAS  Google Scholar 

  16. Min Q, Zhang X, Wu R et al (2011) A novel magnetic mesoporous silica packed S-shaped microfluidic reactor for online proteolysis of low-MW proteome. Chem Commun (Camb) 47:10725–10727. doi:10.1039/c1cc13969j

    Article  CAS  Google Scholar 

  17. Qu H, Wang H, Huang Y et al (2004) Stable microstructured network for protein patterning on a plastic microfluidic channel: strategy and characterization of on-chip enzyme microreactors. Anal Chem 76:6426–6433. doi:10.1021/ac049466g

    Article  CAS  Google Scholar 

  18. Lee J, Musyimi HK, Soper SA, Murray KK (2008) Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS. J Am Soc Mass Spectrom 19:964–972. doi:10.1016/j.jasms.2008.03.015

    Article  CAS  Google Scholar 

  19. Kim BC, Lopez-Ferrer D, Lee S-M et al (2009) Highly stable trypsin-aggregate coatings on polymer nanofibers for repeated protein digestion. Proteomics 9:1893–1900. doi:10.1002/pmic.200800591

    Article  CAS  Google Scholar 

  20. Dodds ED, Seipert RR, Clowers BH et al (2009) Analytical performance of immobilized pronase for glycopeptide footprinting and implications for surpassing reductionist glycoproteomics. J Proteome Res 8:502–512. doi:10.1021/pr800708h

    Article  CAS  Google Scholar 

  21. López-Ferrer D, Hixson KK, Smallwood H et al (2009) Evaluation of a high-intensity focused ultrasound-immobilized trypsin digestion and 18O-labeling method for quantitative proteomics. Anal Chem 81:6272–6277. doi:10.1021/ac802540s

    Article  Google Scholar 

  22. Casadonte F, Pasqua L, Savino R, Terracciano R (2010) Smart trypsin adsorption into N-(2-aminoethyl)-3-aminopropyl-modified mesoporous silica for ultra fast protein digestion. Chemistry 16:8998–9001. doi:10.1002/chem.201000120

    Article  CAS  Google Scholar 

  23. Lim LW, Tomatsu M, Takeuchi T (2006) Development of an on-line immobilized-enzyme reversed-phase HPLC method for protein digestion and peptide separation. Anal Bioanal Chem 386:614–620. doi:10.1007/s00216-006-0458-6

    Article  CAS  Google Scholar 

  24. Guo X, Trudgian DC, Lemoff A et al (2014) Confetti: a multiprotease map of the HeLa proteome for comprehensive proteomics. Mol Cell Proteomics 13:1573–1584. doi:10.1074/mcp.M113.035170

    Article  CAS  Google Scholar 

  25. Cingöz A, Hugon-Chapuis F, Pichon V (2010) Total on-line analysis of a target protein from plasma by immunoextraction, digestion and liquid chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 878:213–221. doi:10.1016/j.jchromb.2009.07.032

    Article  Google Scholar 

  26. Geiser L, Eeltink S, Svec F, Fréchet JMJ (2008) In-line system containing porous polymer monoliths for protein digestion with immobilized pepsin, peptide preconcentration and nano-liquid chromatography separation coupled to electrospray ionization mass spectroscopy. J Chromatogr A 1188:88–96. doi:10.1016/j.chroma.2008.02.075

    Article  CAS  Google Scholar 

  27. Temporini C, Calleri E, Campèse D et al (2007) Chymotrypsin immobilization on epoxy monolithic silica columns: development and characterization of a bioreactor for protein digestion. J Sep Sci 30:3069–3076. doi:10.1002/jssc.200700337

    Article  CAS  Google Scholar 

  28. Yamaguchi H, Miyazaki M, Kawazumi H, Maeda H (2010) Multidigestion in continuous flow tandem protease-immobilized microreactors for proteomic analysis. Anal Biochem 407:12–18. doi:10.1016/j.ab.2010.07.026

    Article  CAS  Google Scholar 

  29. Urban PL, Amantonico A, Zenobi R (2011) Lab-on-a-plate: extending the functionality of MALDI-MS and LDI-MS targets. Mass Spectrom Rev 30:435–478. doi:10.1002/mas.20288

    Article  CAS  Google Scholar 

  30. Jiang B, Yang K, Zhang L et al (2014) Dendrimer-grafted graphene oxide nanosheets as novel support for trypsin immobilization to achieve fast on-plate digestion of proteins. Talanta 122:278–284. doi:10.1016/j.talanta.2014.01.056

    Article  CAS  Google Scholar 

  31. Li Y, Yan B, Deng C et al (2007) On-plate digestion of proteins using novel trypsin-immobilized magnetic nanospheres for MALDI-TOF-MS analysis. Proteomics 7:3661–3671. doi:10.1002/pmic.200700464

    Article  CAS  Google Scholar 

  32. De Stefano L, Rea I, Rendina I et al (2011) Organic–inorganic interfaces for a new generation of hybrid biosensors. Biosens Health Environ Biosecurity

  33. De Stefano L, Rea I, De Tommasi E et al (2009) Bioactive modification of silicon surface using self-assembled hydrophobins from Pleurotus ostreatus. Eur Phys J E Soft Matter 30:181–185. doi:10.1140/epje/i2009-10481-y

    Article  Google Scholar 

  34. Linder MB (2009) Hydrophobins: proteins that self assemble at interfaces. Curr Opin Colloid Interface Sci 14:356–363. doi:10.1016/j.cocis.2009.04.001

    Article  CAS  Google Scholar 

  35. Hektor HJ, Scholtmeijer K (2005) Hydrophobins: proteins with potential. Curr Opin Biotechnol 16:434–439. doi:10.1016/j.copbio.2005.05.004

    Article  CAS  Google Scholar 

  36. Zampieri F, Wösten HAB, Scholtmeijer K (2010) Creating surface properties using a palette of hydrophobins. Materials (Basel) 3:4607–4625. doi:10.3390/ma3094607

    Article  CAS  Google Scholar 

  37. Wohlleben W, Subkowski T, Bollschweiler C et al (2010) Recombinantly produced hydrophobins from fungal analogues as highly surface-active performance proteins. Eur Biophys J 39:457–468. doi:10.1007/s00249-009-0430-4

    Article  CAS  Google Scholar 

  38. Sun T, Qing G, Su B, Jiang L (2011) Functional biointerface materials inspired from nature. Chem Soc Rev 40:2909–2921. doi:10.1039/c0cs00124d

    Article  CAS  Google Scholar 

  39. Qin M, Wang L-K, Feng X-Z et al (2007) Bioactive surface modification of mica and poly(dimethylsiloxane) with hydrophobins for protein immobilization. Langmuir 23:4465–4471. doi:10.1021/la062744h

    Article  CAS  Google Scholar 

  40. Wang Z, Lienemann M, Qiau M, Linder MB (2010) Mechanisms of protein adhesion on surface. Films Hydrophobin 57:8491–8496. doi:10.1021/la101240e

    Google Scholar 

  41. Zhao Z-X, Wang H-C, Qin X et al (2009) Self-assembled film of hydrophobins on gold surfaces and its application to electrochemical biosensing. Colloids Surf B Biointerfaces 71:102–106. doi:10.1016/j.colsurfb.2009.01.011

    Article  CAS  Google Scholar 

  42. Armenante A, Longobardi S, Rea I et al (2010) The Pleurotus ostreatus hydrophobin Vmh2 and its interaction with glucans. Glycobiology 20:594–602

    Article  CAS  Google Scholar 

  43. Longobardi S, Gravagnuolo AM, Rea I et al (2014) Hydrophobin-coated plates as matrix-assisted laser desorption/ionization sample support for peptide/protein analysis. Anal Biochem 449:9–16

    Article  CAS  Google Scholar 

  44. Weng Y, Qu Y, Jiang H et al (2014) An integrated sample pretreatment platform for quantitative N-glycoproteome analysis with combination of on-line glycopeptide enrichment, deglycosylation and dimethyl labeling. Anal Chim Acta 833:1–8. doi:10.1016/j.aca.2014.04.037

    Article  CAS  Google Scholar 

  45. Krenkova J, Szekrenyes A, Keresztessy Z et al (2013) Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis. J Chromatogr A 1322:54–61. doi:10.1016/j.chroma.2013.10.087

    Article  CAS  Google Scholar 

  46. Giardina P, Aurilia V, Cannio R et al (1996) The gene, protein and glycan structures of laccase from Pleurotus ostreatus. Eur J Biochem 235:508–515

    Article  CAS  Google Scholar 

  47. Luo B, Groenke K, Takors R et al (2007) Simultaneous determination of multiple intracellular metabolites in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle by liquid chromatography-mass spectrometry. J Chromatogr A 1147:153–164. doi:10.1016/j.chroma.2007.02.034

    Article  CAS  Google Scholar 

  48. Cooper MA, Singleton VT (2001) A survey of the 2001 to 2005 quartz crystal microbalance biosensor literature: applications of acoustic physics to the analysis of biomolecular interactions. J Mol Recognit 20:154–184. doi:10.1002/jmr.826

    Article  Google Scholar 

  49. Sauerbrey SG (1959) Verwendung von Schwingquarzen zur Wägung dünner Schichten und zur Mikrowägung. Z Phys 155:206–222

    Article  CAS  Google Scholar 

  50. Kannan K, Jasra RV (2009) Immobilization of alkaline serine endopeptidase from Bacillus licheniformis on SBA-15 and MCF by surface covalent binding. J Mol Catal B: Enzym 56:34–40. doi:10.1016/j.molcatb.2008.04.007

    Article  CAS  Google Scholar 

  51. Li S, Wu Z, Lu M et al (2013) Improvement of the enzyme performance of trypsin via adsorption in mesoporous silica SBA-15: hydrolysis of BAPNA. Molecules 18:1138–1149. doi:10.3390/molecules18011138

    Article  CAS  Google Scholar 

  52. Han G, Ye M, Jiang X et al (2009) Comprehensive and reliable phosphorylation site mapping of individual phosphoproteins by combination of multiple stage mass spectrometric analysis with a target-decoy database search. Anal Chem 81:5794–5805. doi:10.1021/ac900702g

    Article  CAS  Google Scholar 

  53. Egrie JC, Browne JK (2001) Development and characterization of novel erythropoiesis stimulating protein (NESP). Br J Cancer 84(Suppl 1):3–10. doi:10.1054/bjoc.2001.1746

    Article  CAS  Google Scholar 

  54. Llop E, Gallego RG, Belalcazar V et al (2007) Evaluation of protein N-glycosylation in 2-DE: erythropoietin as a study case. Proteomics 7:4278–4291. doi:10.1002/pmic.200700572

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by grant from the Ministero dell’Università e della Ricerca Scientifica-Industrial Research Project “Integrated agro-industrial chains with high energy efficiency for the development of eco-compatible processes of energy and biochemicals production from renewable sources and for the land valorization (EnerbioChem)” PON01_01966, funded in the frame of Operative National Programme Research and Competitiveness 2007–2013 D. D. Prot. n. 01/Ric. 18.1.2010.

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Authors and Affiliations

  1. Department of Chemical Sciences, University of Naples “Federico II”, Via Cintia 4, 80126, Naples, Italy

    Sara Longobardi, Alfredo Maria Gravagnuolo, Francesca Pane, Eugenio Galano, Angela Amoresano, Gennaro Marino & Paola Giardina

  2. Department of Physics, University of Naples “Federico II”, Via Cintia 4, 80126, Naples, Italy

    Riccardo Funari & Bartolomeo Della Ventura

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  1. Sara Longobardi
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  2. Alfredo Maria Gravagnuolo
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  3. Riccardo Funari
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  4. Bartolomeo Della Ventura
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  6. Eugenio Galano
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  9. Paola Giardina
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Correspondence to Sara Longobardi.

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This article is dedicated to the memory of Prof. Alessandro Ballio, a Master of Life and Science.

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Longobardi, S., Gravagnuolo, A.M., Funari, R. et al. A simple MALDI plate functionalization by Vmh2 hydrophobin for serial multi-enzymatic protein digestions. Anal Bioanal Chem 407, 487–496 (2015). https://doi.org/10.1007/s00216-014-8309-3

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