1 / 45

Membrane Specific Proteomics

Membrane Specific Proteomics. Martin R. Larsen. Protein Research Group Dept. of Biochemistry and Molecular Biology University of Southern Denmark Odense, Denmark E-mail: mrl@bmb.sdu.dk. Nucleus. Endoplasmic Reticulum. Cell membrane. V acuole. Mitochondrion. Peroxisomes. Lysosomes.

nora
Download Presentation

Membrane Specific Proteomics

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Membrane Specific Proteomics Martin R. Larsen Protein Research Group Dept. of Biochemistry and Molecular Biology University of Southern Denmark Odense, Denmark E-mail: mrl@bmb.sdu.dk

  2. Nucleus Endoplasmic Reticulum Cell membrane Vacuole Mitochondrion Peroxisomes Lysosomes Secretory vesicles Golgi Membrane proteins make up at least one third of total proteins in the cell

  3. Membrane proteins Function: - Inter-/intra-cellular communication - Cellular attachment - Maintenance of the cell membrane potential - Mediation of the transport of ions and proteins - Regulation of vesicle transport - etc……. Transmembrane proteins (1-13 membrane spanning regions) Peripheral proteins (Loosely attached to the membrane e.g., via lipid anchors) Because of the amphipathic structures, membrane proteins are notoriously difficult to handle by any technique

  4. Proteomic´s strategies 1. Traditional (European) 2. Modern (American) 3. GeLC-MS Protein mixture Protein mixture Proteolytic digestion 2 D PAGE Protein mixture Liquid chromatography SDS-PAGE Differential display Mass spectrometry Band excision Spot excision Peptide fragmentation Liquid chromatography Mass spectrometry Database searching Protein identification by MSor MSMS Peptide fragmentation Database searching The big question: ”2D or not 2D” or “LC or not LC”

  5. Protein solubilization for 2DE Chaotropes (protein unfolding/denaturation) - Urea (5 - 9.5 M) - Thiourea (2 M) Surfactants - CHAPS - NP-40 - Triton X-100 - Sulphobetaines (e.g. SB 3-10, ASB14, C8Ø) Bonds/forces within a protein or between proteins • Non-covalent: • Hydrogen bonds • Ionic bonds • Hydrophobic interaction • Van der Waals forces • Covalent: • -Disulfide bonds (C-C) Reducing agents - DTT - DTE - Tributyl phosphine (TBP) Santoni V., electrophoresis 2000 Molloy M., electrophoresis 2000 Highest resolution obtained with 2-DE is under denaturing conditions!

  6. Use of different IEF buffer conditions to fractionate proteins with increasing hydrophobicity. Stronger detergents e.g. ASB14 Or SDS-PAGE Reagent 1: 40 mM Tris Reagent 2: 8 M urea, 4% CHAPS, 40 mM Tris, 0.2% Bio-Lyte 3/10 ampholyte, 2 mM TBP Reagent 3: 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3–10, 40 mM Tris, and 0.2% Bio-Lyte 3/10 ampholyte

  7. A: Normal sample solution B-D: BioRad sequential extraction kit (extraction 1-3)

  8. Simple protocols for purifying membranes/membrane proteins prior to IEF solubilization Triton X100 stripping: Strip cytosolic proteins from the plasma membrane (PM) Triton X100 Supernatant (soluble proteins) Pellet (membrane proteins) Centrifugation Triton X114 (phase partitioning) Triton X114 Upper phase – soluble proteins lower phase – insoluble proteins (detergent phase) Centrifugation Carbonate treatment: Closed vesicles are converted to open membrane sheets, andcontent proteins and loosely attached proteins are released in solubleform. Supernatant (soluble proteins) Sample sonicated in 40 mM Tris (pH 11) Sodium carbonate Incubation on ice (10  excess) Centrifugation (90 min at 15000 g) Pellet (membrane proteins)

  9. Cells treated with the different protocols Followed by SDS PAGE and immunoblotting TX114 Lower phase TX114 Upper phase Insoluble in TX100 Sodium carbonate Soluble in TX100 Total membrane • Santoni V, Kieffer S, Desclaux D, Masson F, Rabilloud T.Electrophoresis 2000 Oct;21(16):3329-44

  10. Different zwitterionic surfactants (”detergents”) used in 2D membrane proteomics. Conventional 2D electrophoresis BioRad reagens 3 Calbiochem Calbiochem

  11. Insoluble fraction from TX100 • Santoni V, Kieffer S, Desclaux D, Masson F, • Rabilloud T.Electrophoresis 2000 Oct;21(16):3329-44

  12. Sample taken from the sodium carbonate extraction • Santoni V, Kieffer S, Desclaux D, Masson F, Rabilloud T.Electrophoresis 2000 Oct;21(16):3329-44

  13. Cell pellet Lysis in 40 mM Tris buffer, pH 11 Incubation in 0.1 M ice-cold sodium carbonate Centrifugation at 15000 g Wash pellet in 40 mM Tris buffer, pH 11 Solubilization in sample solution Proteome analysis of the Pseudomonas aeruginosa, PAO1, Membrane proteome - Nouwens AS et al., Electrophoresis 2000 Nov;21(17):3797-809

  14. Conventional sample solution: 7M Urea, 2M Thiourea, 2% CHAPS, 2% SB 3-10, 2mM TBP, 0,5% carrier ampholytes Optimized sample solution: 7M Urea, 2M Thiourea, 2% CHAPS, 2% SB 3-10 1% ASB 14, 2mM TBP, 0,5% carrier ampholytes

  15. Protein identification Based on MALDI MS: Number of previously characterized proteins: 70 Number of proteins which have significant homology to known membrane proteins from other organisms: 88 ”New” proteins unique To Pseudomonas aeruginosa, PAO1: 30 Approximately 300-350 proteins could be resolved on the gel using Coomassie blue 220 spots were excised form the gel for MS analysis

  16. Differences between normal and optimized sample solubilization 2 1 • Outer membrane porin GRAVY –0,418 • Hydroxamate-type ferrisiderophore receptor GRAVY –0,574 GRAWY: Grand average hydropathy value (a measure of the overall protein hydrophobicity) The higher the GRAVY value the more hydrophobic is the protein !! The ability to solubilize a protein does NOT depend on the overall hydrophobicity of the protein – it is more likely that specific regions within the protein effect the solubilization!!

  17. Characterization of the membrane proteome from sorted X- and Y- Chromosome bearing sperm cells Background: Currently, the X and Y chromosome-bearing sperm that determines sex at fertilization as female and male, respectively, is sorted by using a modified flow cytometer. The method is relatively inefficient for sorting high amounts of sperm cells, resulting in an insufficient number of the sperm cells being successfully sexed. Aim:  Localization and identification of putative sex-specific membrane proteins which could result in the development of new more efficient methods to isolate X and Y chromosome-bearing sperm cells which are of key interest for livestock producers by enabling them to choose the sex of offspring.

  18. 3  108 unsorted pig sperm cells Sodium carbonate extraction solubilization 7M Urea, 2M Thiourea, 1% ASB 14, 2mM TBP, 0,5% carrier ampholytes 7M Urea, 2M Thiourea, 2% CHAPS, 2% SB 3-10, 1% ASB 14, 2mM TBP, 0,5% carrier ampholytes pH 4 pH 4 pH 7 pH 7 pH 4 pH 4 pH 7 pH 7 70 KDa 10 KDa Optimerization of IEF sample buffer

  19. Optimization of the Na2CO3 extraction with respect to starting material ´ ´ ´ ´ 8 8 8 8 3 3 10 10 cells cells 6 6 10 10 cells cells Indicate cytoplasmic proteins ! Marker proteins Marker proteins – – – – membrane associated membrane associated

  20. R1 Dead cells Cell sorting Pig sperm X and Y, Chromosomal difference: 3.5 %

  21. Sorted Pig sperm Pig Y Pig X 5 4 6 11 7 8 9 10 3 2 1 Marked spots differ in expression with > 1.7

  22. Considering 95 % purity in the cell sorting no differences could be detected on the 2 D gels, which could provide basis for discrimination between X and Y in the cell sorting. 22 Proteins showed expression changes > 1.7 - 11 of those could be identified by MALDI tandem mass spectrometry

  23. KDa 90 30 Total number of proteins detected on the gel. 300 pH 10 3 Total number of protein spots tried identified from the 2 D gel. 118 Number of spot where the protein is identified. 52 The majority of the proteins identified using the 2DE approach were membrane proteins derived from either the mitochondrial membrane or the sperm tail. Number of protein spots on the gel with ID protein located to the mitochondria. 35 Number of protein spots on the gel with ID protein located to the sperm tail. 12 Number of protein spots on the gel with ID protein located to the endoplasmatic reticulum. 5 Overview of the 2 D gel experiment

  24. Shaving membranes Limited proteolysis Whole Cell LC-MSMS Enriched Membrane sample LC-MSMS Christine C. Wu and John R. Yates, III. Nature biotechnology 21, 262-267

  25. Liquid chromatography tandem mass spectrometry – membrane shaving 2 100 3: TOF MSMS ES+ % 0 100 2: TOF MSMS ES+ 1 % 0 100 1: TOF MS ES+ % 0 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 Time Limited tryptic proteolysis of sorted cells Reversed-phase capillary LC-MS

  26. Overviews of the LC-MS analysis

  27. Advantages Advantages • Separation of protein isoforms (modifications) • More reliable protein identifications! • High sequence coverage • Sensitive (below pmol level) • Identification of ”difficult” proteins • Quantitation possible – e.g. ICAT/Stable isotope labeling Disadvantages Disadvantages • Large amount of data • Difficult to detect post-translational modifications • Impossible to detect mutations • The majority of proteins are identified based on only one single peptide. • Poor detection of low abundant proteins • Poor detection of basic proteins • Need more material than LC-MSMS • Quantitation can be relatively hard The two techniques are highly complementary 2 DE LC-MSMS Overall the two techniques complements each other

  28. Example: Diabetes type 1 Diabetes Mellitus (DM) is a group of disorders characterized by chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action or both. Pancreas Langerhansk ø ß-celle Muskelfiber Blodkar Insulin Glucose T1D: Absolute insulin deficiency due to an autoimmune associated destruction of the insulin producing ß-cells in the islets of Langerhans. T2D: Relative insulin deficiency due to decreased effect of insulin in the target tissues e.g. muscles and adipose tissue (insulin resistance) or due to a secretory defect of insulin with or without insulin resistance.

  29. Copenhagen Model Nerup, J. Et al., 1994, Diabetologia37 (Suppl 2), S82-S89. Klonal T- and B-lymfocyt ekspansion IL-1 ? Virus ? Chemicals ? IL-1 Nutrition ? • • • • • • • • • • • • • Mø a • TNF • • • • • • Beta + cells Th lymphocyte O - g IFN IL-1 2 NO • • Mø / EC • • • • • • O - • • • 2 NO Cytokines => inducible nitric oxide synthase expression => NO generation

  30. Investigation of the effect of Interleukin-1 on isolated islets Islet isolation S35-methionine IL-1ß + S35-methionine SDS PAGE Mass spectrometric protein identification pH Cytokines => iNOS expression => NO generation => -cell destruction L-Arginine analogs (NMMA) H. U. Andersen et al, Electrophoresis 1997 P. Mose Larsen et al, Diabetes, 2001

  31. The effect of interleukine 1 on the plasma membrane sub-proteome of insulin producing beta cells Plasma membrane • Plasma membrane proteins constitute only 3-5 % of the whole proteome. • Cell-cell communication/attachment • Signalling. • Transport of ions and proteins. • First barrier to the environment • Important potential drug targets and markers for the pharmaceutical industry Important in Diabetes

  32. Plasma membrane sub-proteome - strategy SILAC: Stable isotop labeling with isotop amino acids Cell line in media containing Lys(13C) + Arg(13C,15N) Cell line in normal amino acid media  IL - 1 + IL - 1 Reference Experiment 1 Trypsin digestion Pool 1:1 TiO2 purification Cell lysis - mechanical in succrose buffer Bound - phosphopeptides Flowthrough Succrose centrifugation and Na2CO3 treatment LC-MSMS LC-MSMS Centrifugation Solubilization using SDS sample buffer SDS - PAGE

  33. Tumor-associated calcium signal transducer 1 [Rattus norvegicus] EMGEIHR 5 Da GESLFHSSK 3 Da Plasma membranes In-solution trypsin digestion Strong cation exchange LC-MSMS 10 mM KCL fraction

  34. Cation-independent mannose-6-phosphate receptor LVSFHDDSDEDLLHI.- C-terminal peptide from protein – can not be quantified by SILAC L+H

  35. Golgi vesicular membrane trafficking protein p18 SLSIEIGHEVK L H

  36. Glycosyl-phosphatidylinositol anchored proteins: Membrane attached surface proteins GPI-anchored protein NH2 C terminus • Widespread class of membrane proteins • Acts as • enzymes • adhesion molecules • receptors • antigens • … Ethanolamine Man Man P Ins GlcN Man Exterior P DAG Plasma membrane Interior Ferguson et al. (1988) Science 239, 753-759

  37. Strategy for isolation and identification of GPI-anchored proteins PI-PLC LC-MSMS Elortza, F et al, 2004 Cell culture Isolation of membrane fraction • Isolation of GPI proteins • PI-PLC treatment • Two-phase separation SDS-PAGE of extracted protein Identification of GPI-proteins by nanoLC-MS/MS

  38. Identification of proteins in human lipid rafts + PI-PLC Untreated 10 slices Trypsin DB search 16 proteins LC-MS/MS Literature Sequence analysis 6 GPI-anchored proteins

  39. Human GPI-proteins (lipid rafts) • Accession no. # peptides • Alkaline phosphatase P05186 11 • Decay acceleration factor P08174 9 • Folate receptor 1 P15328 8 • CD59 glycoprotein P13987 3 • Carboxypeptidase P14284 2 • Urokinase plasminogen activator receptor Q03405 1 OTHER: Fibronectin receptor-CD29 (P05556), Galactoprotein-CD49c (P26006), Melanoma adhesion molecule-CD146 (P431214), F2 heavy chain antigen-CD98 (P08195), Epican-CD44 (P16070), 78 kDa glucose-regulated protein (P11021), Mesotheline/megakaryocyte potentiation factor (Q9UK57), Actin / actin (P02571/P02572), Voltage dependent anion channel (P21796), B-cell antigen receptor complex associated protein alpha-chain-CD79 (P11912).

  40. GPI-proteins in A. thaliana • Genome: ~ 25.000 ORFs • Prediction: 210 GPI-APs (Borner et al., 2002) • Only few GPI-APs have been experimentally verified • Good test case for our strategy Collaborators: T. Nühse and Scott Peck, Norwich, UK.

  41. Isolation of GPI-anchored proteins from A. thaliana cell line

  42. Identification of GPI-anchored proteins in A. thaliana byLC-MS/MS (Q-TOF) • 64 protein identified in 16 gel slices: • 44 bona fide GPI-anchored proteins validated by sequence analysis and prediction tools • 20 proteins with TM domains or secretion signals, i.e. non-GPI-APs

  43. A few other ways to look at membrane proteins • Top-down proteomics. HPLC separation of membrane proteins in high concentration of formic acid. Has to use FT-MS instrumentation • Labeling of the plasma membranes using biotin derivatives that are not membrane permeable. Problem: the biotin derivatives are almost always permeable leading to purification of non-membrane proteins. • We are working on alternative methods for purification of especially plasma membrane proteins………..

  44. Conclusion  Membrane proteins can be efficiently separated by 2 DEusing strong detergents in the IEF buffer.  Sodium carbonate treatment or high salt treatment is crucial for elimination of non-membrane proteins – cytoplasmic contaminants…  Surface membrane proteins can be efficiently identified using LC-MSMS of peptides derived from proteolysis of intact cells or enriched membrane fractions. However, cell sorting is crucial here….  2DE and LC-MSMS is highly complementary for any proteomic work.

  45. Acknowledgements University of Southern Denmark Peter Roepstorff Ole N. Jensen Felix Elortza Australian Proteome Analysis Facility Stuart Cordwell Brad Walsh Derek Van Dyk Amanda Nouwens Faculty of Veterinary Science Gareth Evans Bengt Eriksson

More Related