JoVE Journal > Biochemistry
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
생화학

Identifikation af inositol fosfat eller Phosphoinositide interagerende proteiner ved affinitets kromatografi koblet til Western blot eller massespektrometri

Published: July 26, 2019
doi:
10.3791/59865
1Institute of Parasitology,McGill University

Summary

Denne protokol fokuserer på identifikation af proteiner, der binder sig til inositol phosphater eller phosphoinositides. Det bruger affinitets kromatografi med biotinylerede inositol phosphater eller phosphoinositides, der er immobiliseret via streptavidin til agopstået eller magnetiske perler. Inositol fosfat eller phosphoinositide binding proteiner er identificeret ved Western blotting eller massespektrometri.

Abstract

Inositol phosphater og phosphoinositides regulere flere cellulære processer i eukaryoter, herunder genekspression, vesikelprotein handel, signaltransduktion, metabolisme, og udvikling. Disse metabolitter udfører denne regulerings aktivitet ved at binde sig til proteiner og derved ændre protein konformationen, katalytisk aktivitet og/eller interaktioner. Den beskrevne metode bruger affinitets kromatografi koblet til massespektrometri eller Western blotting til at identificere proteiner, der interagerer med inositol phosphater eller phosphoinositides. Inositol phosphater eller phosphoinositides er kemisk mærket med biotin, som derefter fanges via streptavidin konjugeret til agopstået eller magnetiske perler. Proteiner isoleres ved deres affinitet af binding til metabolitten, derefter elueres og identificeres ved massespektrometri eller Western blotting. Metoden har en enkel arbejdsgang, der er følsom, ikke-radioaktiv, liposome-fri, og tilpasselig, støtte analyse af protein og metabolit interaktion med præcision. Denne fremgangsmåde kan anvendes i label-fri eller i aminosyre-mærkede kvantitative massespektrometri metoder til at identificere protein-metabolit interaktioner i komplekse biologiske prøver eller ved hjælp af rensede proteiner. Denne protokol er optimeret til analyse af proteiner fra Trypanosoma brucei, men det kan tilpasses til beslægtede protozoer parasitter, gær eller pattedyrceller.

Introduction

Inositol phosphater (IPS) og phosphoinositides (pi’er) spiller en central rolle i eukaryote biologi gennem regulering af cellulære processer såsom kontrol af genekspression1,2,3, vesikle handel 4, signaltransduktion5,6, metabolisme7,8,9, og udvikling8,10. Den regulerende funktion af disse metabolitter skyldes deres evne til at interagere med proteiner og dermed regulere protein funktion. Ved binding af proteiner kan IPs og Pi’er ændre protein konformationen11, katalytisk aktivitet12eller interaktioner13 og dermed påvirke cellulær funktion. IPS og pi’er distribueres i flere subcellulære rum, såsom Nucleus2,3,14,15, endoplasmatiske reticulum16,17, plasma membran1 og cytosol18, enten associeret med proteiner3,19 eller med RNAs20.

Spaltningen af membranen-associeret PI (4,5) P2 ved fosfolipase C resulterer i frigivelse af ins (1, 4, 5) P3, som kan fosforyleres eller defosforyleres ved henholdsvis IP-kinaser og fosfataser. IPs er opløselige molekyler, der kan binde sig til proteiner og udøve regulerende funktioner. For eksempel, ins (1, 4, 5) P3 i metazoan kan fungere som en anden budbringer ved binding til IP3 receptorer, som inducerer receptoren konformationsmæssige ændringer og dermed frigivelse af ca2 + fra intracellulære butikker11. Ins (1, 3, 4, 5) P4 binder sig til Histon deacetylase-komplekset og regulerer protein kompleks montage og aktivitet13. Andre eksempler på IPS reguleringsfunktion omfatter kontrol af kromatin organisation21, RNA transport22,23, RNA redigering24, og transkriptionen1,2,3 . I modsætning hertil er pi’er ofte forbundet med rekruttering af proteiner til plasma membranen eller organelle membraner25. Men en ny ejendom af pi’er er evnen til at associere med proteiner i et ikke-membranøs miljø3,15,19,26. Dette er tilfældet med den nukleare receptor akut faktor, som transkriptional kontrolfunktion er reguleret af PI (3, 4, 5) P319, og poly-A polymerase hvilken enzymatisk aktivitet er reguleret af nukleare PI (4,5) P226. En regulerende rolle for IPS og pi’er er blevet vist i mange organismer, herunder gær22,27, pattedyrceller19,23, Drosophila10 og Worms28. Af betydning er disse metabolitters rolle i trypanosomer, som afveg tidligt fra den eukaryotiske afstamning. Disse metabolitter spiller en væsentlig rolle i Trypanosoma brucei transkriptional Control1,3, Development8, organelle Biogenese og protein Traffic29,30 , 31 , 32, og er også involveret i at kontrollere udvikling og infektion i patogener T. cruzi33,34,35, Toxoplasma36 og Plasmodium 5 , 37. derfor kan forståelse af IPS ‘ og pi’s rolle i trypanosomer bidrage til at belyse nye biologiske funktioner for disse molekyler og identificere nye narkotikaprækursorer.

Specificiteten af protein og IP eller PI binding afhænger af protein interagere domæner og fosforylering tilstand af inositol13,38, selv om interaktioner med lipiddelen af pi’er også forekommer19. Variationen af IPs og Pi’er og deres modificerende kinaser og fosfataser giver en fleksibel cellulær mekanisme til kontrol af protein funktion, som påvirkes af metabolitten tilgængelighed og overflod, fosforylering tilstand af inositol, og protein affinitet af interaktion1,3,13,38. Selv om nogle protein domæner er velkarakteriserede39,40,41, fx pleckstrin homologi Domain42 og SPX (SYG1/Pho81/XPR1) domæner43 ,44,45, nogle proteiner interagerer med IPS eller pi’er af mekanismer, der forbliver ukendte. For eksempel mangler den repressor-aktivator protein 1 (RAP1) af T. brucei CANONICAL pi-binding domæner, men INTERAGERER med PI (3, 4, 5) P3 og kontrol transkriptionen af gener involveret i antigen variation3. Affinitets kromatografi og massespektrometri-analyse af IP-eller PI-interagerende proteiner fra trypanosome, gær eller pattedyrsceller identificerede flere proteiner uden kendte IP-eller PI-bindings domæner8,46, 47. dataene tyder på yderligere ukarakteriserede protein domæner, der binder sig til disse metabolitter. Derfor, identifikation af proteiner, der interagerer med IPs eller Pi’er kan afsløre nye mekanismer af protein-metabolit interaktion og nye cellulære regulerende funktioner for disse små molekyler.

Den beskrevne metode beskæftiger sig med affinitets kromatografi koblet til Western blotting eller massespektrometri for at identificere proteiner, der binder sig til IPs eller Pi’er. Det bruger biotinylerede IPs eller Pi’er, der enten er tværbundet til streptavidin konjugeret til aganerede perler eller alternativt, fanget via streptavidin-konjugeret magnetiske perler (figur 1). Metoden giver en enkel arbejdsgang, der er følsom, ikke-radioaktiv, liposome-fri og er egnet til påvisning af binding af proteiner fra celle lysater eller renset proteiner3 (figur 2). Metoden kan anvendes i etiket-fri8,46 eller koblet til aminosyre-mærket kvantitativ massespektrometri47 til at identificere IP eller PI-bindende proteiner fra komplekse biologiske prøver. Derfor er denne metode et alternativ til de få metoder til rådighed til at studere samspillet mellem IPs eller Pi’er med cellulære proteiner og vil hjælpe med at forstå den regulerende funktion af disse metabolitter i trypanosomes og måske andre eukaryoter.

Protocol

1. analyse af IP-eller PI-bindende proteiner ved affinitets kromatografi og Western blotting Cellevækst, lysis og affinitets kromatografi Grow T. brucei celler til mid-log fase og overvåge cellelevedygtighed og tæthed. I alt 5,0 x 107 celler er tilstrækkelig til en bindende analyse. For blodbanen former, dyrke celler i HMI-9 medier suppleret med 10% føtal kvægserum (FBS) ved 37 °C og 5% CO2. Hold celletætheden mellem 8,0 x 105 t…

Representative Results

Analyse af RAP1 og PI (3, 4, 5) P3 interaktion ved affinitets kromatografi og Western blottingDette eksempel illustrerer anvendelsen af denne metode til at analysere bindingen af pi’er ved RAP1 fra t. brucei lysat eller ved rekombinant T. brucei RAP1 protein. Lysates af T. brucei blodbanen former, der udtrykker hemagglutinin (ha)-Tagged RAP1 blev brugt i bindende assays. RAP1 er et protein, der er involveret i transkriptional kontrol af variant overflade glykoprotein (VSG) …

Discussion

Identifikationen af proteiner, der binder sig til IPs eller Pi’er, er afgørende for at forstå disse metabolitters cellulære funktion. Affinitets kromatografi koblet til Western blot eller massespektrometri giver mulighed for at identificere IP-eller PI-interagerende proteiner og dermed få indsigt i deres reguleringsfunktion. IPS eller pi’er, der er kemisk mærkede [f. eks. ins (1, 4, 5) P3 kemisk forbundet med biotin] og tværbundet til aganerede perler via streptavidin eller fanget af streptavidin magnetiske perler …

Disclosures

The authors have nothing to disclose.

Acknowledgements

Dette arbejde blev støttet af Canadas naturvidenskabs-og ingeniør Forskningsråd (NSERC, RGPIN-2019-04658); NSERC Discovery Launch supplement til Early Career forskere (DGECR-2019-00081) og af McGill University.

Materials

Acetone Sigma-Aldrich 650501 Ketone
Acetonitrile Sigma-Aldrich 271004 Solvent 
Ammonium bicarbonate Sigma-Aldrich A6141 Inorganic salt
Centrifuge Avanti J6-MI Beckman Coulter Avanti J6-MI Centrifuge for large volumes (e.g., 1L)
Centrifuge botles Sigma-Aldrich B1408 Bottles for centrifugation of 1L of culture
Control Beads Echelon P-B000-1ml Affinity chromatography reagent – control
D-(+)-Glucose Sigma-Aldrich G8270 Sugar, Added in PBS to keep cells viable
Dithiothreitol (DTT)  Bio-Rad 1610610 Reducing agent
Dynabeads M-270 Streptavidin ThermoFisher Scientific 65305 Streptavidin beads for binding to biotin ligands
EDTA-free Protease Inhibitor Cocktail Roche 11836170001 Protease inhibitors
Electrophoresis running buffer Bio-Rad 1610732 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3
Falcon 15 mL Conical Centrifuge Tubes Corning Life Sciences 430052 To centrifuge 10 mL cultures
Formic acid Sigma-Aldrich 106526 Acid
Glycine Sigma-Aldrich G7126 Amino acid
HMI-9 cell culture medium ThermoFisher Scientific ME110145P1 Cell culture medium for T. brucei bloodstream forms
Imperial Protein Stain ThermoFisher Scientific 24615 Coomassie staining for protein detection in SDS/PAGE
Ins(1,4,5)P3 Beads Echelon Q-B0145-1ml Affinity chromatography reagent 
Instant Nonfat Dry Milk Thomas Scientific C837M64 Blocking reagent for Western blotting
Iodoacetamide Sigma-Aldrich I6125 Alkylating reagent for cysteine proteins or peptides
Lab Rotator Thomas Scientific 1159Z92 For binding assays
LoBind Microcentrifuge Tubes ThermoFisher Scientific 13-698-793 Low protein binding tubes for mass spectrometry
Nonidet P-40 (Igepal CA-630) Sigma-Aldrich 21-3277 Detergent
PBS, pH 7.4 ThermoFisher Scientific 10010031 Physiological buffer
Peroxidase substrate for chemiluminescence ThermoFisher Scientific 32106 Substrate for Western bloting detection of proteins
PhosSTOP Phosphatase Inhibitor Cocktail Tablets Roche 4906845001 Phosphatase inhibitors
PI(3)P PIP Beads Echelon P-B003a-1ml Affinity chromatography reagent 
PI(3,4)P2 PIP Beads Echelon P-B034a-1ml Affinity chromatography reagent 
PI(3,4,5)P3 diC8 Echelon P-3908-1mg Affinity chromatography reagent 
PI(3,4,5)P3 PIP Beads Echelon P-B345a-1ml Affinity chromatography reagent 
PI(3,5)P2 PIP Beads Echelon P-B035a-1ml Affinity chromatography reagent 
PI(4)P PIP Beads Echelon P-B004a-1ml Affinity chromatography reagent 
PI(4,5)P2 diC8 Echelon P-4508-1mg Affinity chromatography reagent 
PI(4,5)P2 PIP Beads Echelon P-B045a-1ml Affinity chromatography reagent 
PI(5)P PIP Beads Echelon P-B005a-1ml Affinity chromatography reagent 
Ponceau S solution Sigma-Aldrich P7170 Protein staining (0.1% [w/v] in 5% acetic acid)
Potassium hexacyanoferrate(III) Sigma-Aldrich 702587 Potassium salt 
PtdIns PIP Beads Echelon P-B001-1ml Affinity chromatography reagent 
PVDF Membrane Bio-Rad 1620177 For Western blotting 
Refrigerated centrifuge Eppendorf 5910 R Microcentrifuge for small volumes (e.g., 1.5 mL)
Sodium dodecyl sulfate Sigma-Aldrich 862010 Detergent
Sodium thiosulfate Sigma-Aldrich 72049 Chemical 
SpeedVac Vacuum Concentrators ThermoFisher Scientific SPD120-115 Sample concentration (e.g., for mass spectrometry)
T175 flasks for cell culture  ThermoFisher Scientific 159910 To grow 50 mL T. brucei culture
Trypsin, Mass Spectrometry Grade Promega V5280 Trypsin for protein digestion
Urea Sigma-Aldrich U5128 Denaturing reagent
Vortex Fisher Scientific 02-215-418 For mixing reactions
Western blotting transfer buffer Bio-Rad 1610734 25 mM Tris, 192 mM glycine, pH 8.3 with 20% methanol
Whatman 3 mm paper Sigma-Aldrich WHA3030861 Paper for Wester transfer
2-mercaptoethanol (14.2 M) Bio-Rad 1610710 Reducing agent
2x Laemmli Sample Buffer Bio-Rad 161-0737 Protein loading buffer
4–20% Mini-PROTEAN TGX Precast Protein Gels Bio-Rad 4561094 Gel for protein electrophoresis
4x Laemmli Sample Buffer Bio-Rad 161-0747 Protein loading buffer
DOWNLOAD MATERIALS LIST

References

  1. Cestari, I., Stuart, K. Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes. Proceedings of the National Academy of Sciences of the United States of America. 112 (21), 2803-2812 (2015).
  2. Odom, A. R., Stahlberg, A., Wente, S. R., York, J. D. A role for nuclear inositol 1,4,5-trisphosphate kinase in transcriptional control. Science. 287 (5460), 2026-2029 (2000).
  3. Cestari, I., McLeland-Wieser, H., Stuart, K. Nuclear Phosphatidylinositol 5-Phosphatase Is Essential for Allelic Exclusion of Variant Surface Glycoprotein Genes in Trypanosomes. Molecular and Cellular Biology. 39 (3), (2019).
  4. Billcliff, P. G., et al. OCRL1 engages with the F-BAR protein pacsin 2 to promote biogenesis of membrane-trafficking intermediates. Molecular Biology of the Cell. 27 (1), 90-107 (2016).
  5. Brochet, M., et al. Phosphoinositide metabolism links cGMP-dependent protein kinase G to essential Ca(2)(+) signals at key decision points in the life cycle of malaria parasites. PLoS Biology. 12 (3), 1001806 (2014).
  6. Azevedo, C., Szijgyarto, Z., Saiardi, A. The signaling role of inositol hexakisphosphate kinases (IP6Ks). Advances in Enzyme Regulation. 51 (1), 74-82 (2011).
  7. Szijgyarto, Z., Garedew, A., Azevedo, C., Saiardi, A. Influence of inositol pyrophosphates on cellular energy dynamics. Science. 334 (6057), 802-805 (2011).
  8. Cestari, I., Anupama, A., Stuart, K. Inositol polyphosphate multikinase regulation of Trypanosoma brucei life stage development. Molecular Biology of the Cell. 29 (9), 1137-1152 (2018).
  9. Bang, S., et al. AMP-activated protein kinase is physiologically regulated by inositol polyphosphate multikinase. Proceedings of the National Academy of Sciences of the United States of America. 109 (2), 616-620 (2012).
  10. Seeds, A. M., Tsui, M. M., Sunu, C., Spana, E. P., York, J. D. Inositol phosphate kinase 2 is required for imaginal disc development in Drosophila. Proceedings of the National Academy of Sciences of the United States of America. 112 (51), 15660-15665 (2015).
  11. Hamada, K., Miyatake, H., Terauchi, A., Mikoshiba, K. IP3-mediated gating mechanism of the IP3 receptor revealed by mutagenesis and X-ray crystallography. Proceedings of the National Academy of Sciences of the United States of America. 114 (18), 4661-4666 (2017).
  12. Watson, P. J., et al. Insights into the activation mechanism of class I HDAC complexes by inositol phosphates. Nature Communications. 7, 11262 (2016).
  13. Watson, P. J., Fairall, L., Santos, G. M., Schwabe, J. W. Structure of HDAC3 bound to co-repressor and inositol tetraphosphate. Nature. 481 (7381), 335-340 (2012).
  14. Irvine, R. F. Nuclear lipid signalling. Nature Reviews: Molecular Cell Biology. 4 (5), 349-360 (2003).
  15. Sobol, M., et al. UBF complexes with phosphatidylinositol 4,5-bisphosphate in nucleolar organizer regions regardless of ongoing RNA polymerase I activity. Nucleus. 4 (6), 478-486 (2013).
  16. Nascimbeni, A. C., et al. ER-plasma membrane contact sites contribute to autophagosome biogenesis by regulation of local PI3P synthesis. EMBO Journal. 36 (14), 2018-2033 (2017).
  17. Martin, K. L., Smith, T. K. The myo-inositol-1-phosphate synthase gene is essential in Trypanosoma brucei. Biochemical Society Transactions. 33, 983-985 (2005).
  18. Matsu-ura, T., et al. Cytosolic inositol 1,4,5-trisphosphate dynamics during intracellular calcium oscillations in living cells. Journal of Cell Biology. 173 (5), 755-765 (2006).
  19. Blind, R. D., et al. The signaling phospholipid PIP3 creates a new interaction surface on the nuclear receptor SF-1. Proceedings of the National Academy of Sciences of the United States of America. 111 (42), 15054-15059 (2014).
  20. Lin, A., et al. The LINK-A lncRNA interacts with PtdIns(3,4,5)P3 to hyperactivate AKT and confer resistance to AKT inhibitors. Nature Cell Biology. 19 (3), 238-251 (2017).
  21. Steger, D. J., Haswell, E. S., Miller, A. L., Wente, S. R., O’Shea, E. K. Regulation of chromatin remodeling by inositol polyphosphates. Science. 299 (5603), 114-116 (2003).
  22. York, J. D., Odom, A. R., Murphy, R., Ives, E. B., Wente, S. R. A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export. Science. 285 (5424), 96-100 (1999).
  23. Adams, R. L., Mason, A. C., Glass, L., Aditi, S. R., Wente, Nup42 and IP6 coordinate Gle1 stimulation of Dbp5/DDX19B for mRNA export in yeast and human cells. Traffic. 18 (12), 776-790 (2017).
  24. Macbeth, M. R., et al. Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing. Science. 309 (5740), 1534-1539 (2005).
  25. Dickson, E. J., Hille, B. Understanding phosphoinositides: rare, dynamic, and essential membrane phospholipids. Biochemical Journal. 476 (1), 1-23 (2019).
  26. Mellman, D. L., et al. A PtdIns4,5P2-regulated nuclear poly(A) polymerase controls expression of select mRNAs. Nature. 451 (7181), 1013-1017 (2008).
  27. Lee, Y. S., Mulugu, S., York, J. D., O’Shea, E. K. Regulation of a cyclin-CDK-CDK inhibitor complex by inositol pyrophosphates. Science. 316 (5821), 109-112 (2007).
  28. Nagy, A. I., et al. IP3 signalling regulates exogenous RNAi in Caenorhabditis elegans. EMBO Reports. 16 (3), 341-350 (2015).
  29. Hall, B. S., et al. TbVps34, the trypanosome orthologue of Vps34, is required for Golgi complex segregation. Journal of Biological Chemistry. 281 (37), 27600-27612 (2006).
  30. Rodgers, M. J., Albanesi, J. P., Phillips, M. A. Phosphatidylinositol 4-kinase III-beta is required for Golgi maintenance and cytokinesis in Trypanosoma brucei. Eukaryotic Cell. 6 (7), 1108-1118 (2007).
  31. Gilden, J. K., et al. The role of the PI(3,5)P2 kinase TbFab1 in endo/lysosomal trafficking in Trypanosoma brucei. Molecular and Biochemical Parasitology. 214, 52-61 (2017).
  32. Demmel, L., et al. The endocytic activity of the flagellar pocket in Trypanosoma brucei is regulated by an adjacent phosphatidylinositol phosphate kinase. Journal of Cell Science. 129 (11), 2285 (2016).
  33. Gimenez, A. M., et al. Phosphatidylinositol kinase activities in Trypanosoma cruzi epimastigotes. Molecular and Biochemical Parasitology. 203 (1-2), 14-24 (2015).
  34. Hashimoto, M., et al. Inositol 1,4,5-trisphosphate receptor regulates replication, differentiation, infectivity and virulence of the parasitic protist Trypanosoma cruzi. Molecular Microbiology. 87 (6), 1133-1150 (2013).
  35. Cestari, I., Haas, P., Moretti, N. S., Schenkman, S., Stuart, K. Chemogenetic Characterization of Inositol Phosphate Metabolic Pathway Reveals Druggable Enzymes for Targeting Kinetoplastid Parasites. Cell Chemical Biology. 23 (5), 608-617 (2016).
  36. Lovett, J. L., Marchesini, N., Moreno, S. N., Sibley, L. D. Toxoplasma gondii microneme secretion involves intracellular Ca(2+) release from inositol 1,4,5-triphosphate (IP(3))/ryanodine-sensitive stores. Journal of Biological Chemistry. 277 (29), 25870-25876 (2002).
  37. McNamara, C. W., et al. Targeting Plasmodium PI(4)K to eliminate malaria. Nature. 504 (7479), 248-253 (2013).
  38. Tresaugues, L., et al. Structural basis for phosphoinositide substrate recognition, catalysis, and membrane interactions in human inositol polyphosphate 5-phosphatases. Structure. 22 (5), 744-755 (2014).
  39. Varnai, P., et al. Quantifying lipid changes in various membrane compartments using lipid binding protein domains. Cell Calcium. 64, 72-82 (2017).
  40. Lystad, A. H., Simonsen, A. Phosphoinositide-binding proteins in autophagy. FEBS Letters. 590 (15), 2454-2468 (2016).
  41. Cullen, P. J., Cozier, G. E., Banting, G., Mellor, H. Modular phosphoinositide-binding domains–their role in signalling and membrane trafficking. Current Biology. 11 (21), 882-893 (2001).
  42. Klarlund, J. K., et al. Signaling by phosphoinositide-3,4,5-trisphosphate through proteins containing pleckstrin and Sec7 homology domains. Science. 275 (5308), 1927-1930 (1997).
  43. Wild, R., et al. Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains. Science. 352 (6288), 986-990 (2016).
  44. Gerasimaite, R., et al. Inositol Pyrophosphate Specificity of the SPX-Dependent Polyphosphate Polymerase VTC. ACS Chemical Biology. 12 (3), 648-653 (2017).
  45. Potapenko, E., et al. 5-Diphosphoinositol pentakisphosphate (5-IP7) regulates phosphate release from acidocalcisomes and yeast vacuoles. Journal of Biological Chemistry. 293 (49), 19101-19112 (2018).
  46. Wu, M., Chong, L. S., Perlman, D. H., Resnick, A. C., Fiedler, D. Inositol polyphosphates intersect with signaling and metabolic networks via two distinct mechanisms. Proceedings of the National Academy of Sciences of the United States of America. 113 (44), 6757-6765 (2016).
  47. Jungmichel, S., et al. Specificity and commonality of the phosphoinositide-binding proteome analyzed by quantitative mass spectrometry. Cell Reports. 6 (3), 578-591 (2014).
  48. Yang, X., Figueiredo, L. M., Espinal, A., Okubo, E., Li, B. RAP1 is essential for silencing telomeric variant surface glycoprotein genes in Trypanosoma brucei. Cell. 137 (1), 99-109 (2009).
  49. Cestari, I., Stuart, K. Transcriptional Regulation of Telomeric Expression Sites and Antigenic Variation in Trypanosomes. Current Genomics. 19 (2), 119-132 (2018).
  50. Clough, T., Thaminy, S., Ragg, S., Aebersold, R., Vitek, O. Statistical protein quantification and significance analysis in label-free LC-MS experiments with complex designs. BMC Bioinformatics. 13, 6 (2012).
  51. Schneider, A., Charriere, F., Pusnik, M., Horn, E. K. Isolation of mitochondria from procyclic Trypanosoma brucei. Methods in Molecular Biology. 372, 67-80 (2007).
  52. DeGrasse, J. A., Chait, B. T., Field, M. C., Rout, M. P. High-yield isolation and subcellular proteomic characterization of nuclear and subnuclear structures from trypanosomes. Methods in Molecular Biology. 463, 77-92 (2008).
  53. Opperdoes, F. R. A rapid method for the isolation of intact glycosomes from Trypanosoma brucei by Percoll -gradient centrifugation in a vertical rotor. Molecular and Biochemical Parasitology. 3 (3), 181-186 (1981).
  54. Pereira, N. M., de Souza, W., Machado, R. D., de Castro, F. T. Isolation and properties of flagella of trypanosomatids. The Journal of protozoology. 24 (4), 511-514 (1977).
  55. Subota, I., et al. Proteomic analysis of intact flagella of procyclic Trypanosoma brucei cells identifies novel flagellar proteins with unique sub-localization and dynamics. Molecular and Cellular Proteomics. 13 (7), 1769-1786 (2014).
  56. Fukuda, M., Kojima, T., Kabayama, H., Mikoshiba, K. Mutation of the pleckstrin homology domain of Bruton’s tyrosine kinase in immunodeficiency impaired inositol 1,3,4,5-tetrakisphosphate binding capacity. Journal of Biological Chemistry. 271 (48), 30303-30306 (1996).
  57. Knodler, A., Mayinger, P. Analysis of phosphoinositide-binding proteins using liposomes as an affinity matrix. BioTechniques. 38 (6), (2005).
  58. Best, M. D. Global approaches for the elucidation of phosphoinositide-binding proteins. Chemistry and Physics of Lipids. 182, 19-28 (2014).

Tags

Inositol PhosphatePhosphoinositideProtein BindingAffinity ChromatographyWestern BlotMass SpectrometryRegulatory FunctionSignal TransductionCell DevelopmentBiotinylatedLyposome FreeT. BruceiPBS-GLysis BufferProtease InhibitorPhosphatase InhibitorBinding Assay
check_url/kr/59865?article_type=t&slug=identification-inositol-phosphate-or-phosphoinositide-interacting

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Cite This Article
Cestari, I. Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry. J. Vis. Exp. (149), e59865, doi:10.3791/59865 (2019).
Download Citation
Reprints and Permissions

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image