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Membrane Specific Proteomics Martin R. Larsen Protein Research Group Dept. of Biochemistry and Molecular Biology University of Southern Denmark Odense, Denmark E-mail: [email protected] Membrane proteins make up at least one third of total proteins in the cell Nucleus Endoplasmic Reticulum Cell membrane Vacuole Mitochondrion Peroxisomes Lysosomes Golgi Secretory vesicles 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 Proteomic´s strategies 1. Traditional (European) Protein mixture 3. GeLC-MS Protein mixture SDS-PAGE 2 D PAGE Band excision Differential display Liquid chromatography Mass spectrometry Spot excision 2. Modern (American) Protein mixture Proteolytic digestion Liquid chromatography Mass spectrometry Peptide fragmentation Peptide fragmentation Protein identification by MS or MSMS Database searching Database searching The big question: or ”2D or not 2D” “LC or not LC” Protein solubilization for 2DE Highest resolution obtained with 2-DE is under denaturing conditions! 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) Reducing agents - DTT - DTE - Tributyl phosphine (TBP) 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) Santoni V., electrophoresis 2000 Molloy M., electrophoresis 2000 Use of different IEF buffer conditions to fractionate proteins with increasing hydrophobicity. 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 Stronger detergents e.g. ASB14 Or SDS-PAGE A: Normal sample solution B-D: BioRad sequential extraction kit (extraction 1-3) 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 Centrifugation lower phase – insoluble proteins (detergent phase) Carbonate treatment: Closed vesicles are converted to open membrane sheets, and content proteins and loosely attached proteins are released in soluble form. Sample sonicated in 40 mM Tris (pH 11) Supernatant (soluble proteins) Sodium carbonate Incubation on ice (10 excess) Centrifugation (90 min at 15000 g) Pellet (membrane proteins) Sodium carbonate TX114 Lower phase TX114 Upper phase Insoluble in TX100 Soluble in TX100 Total membrane Cells treated with the different protocols Followed by SDS PAGE and immunoblotting Santoni V, Kieffer S, Desclaux D, Masson F, Rabilloud T. Electrophoresis 2000 Oct;21(16):3329-44 Different zwitterionic surfactants (”detergents”) used in 2D membrane proteomics. Conventional 2D electrophoresis BioRad reagens 3 Calbiochem Calbiochem Insoluble fraction from TX100 Santoni V, Kieffer S, Desclaux D, Masson F, Rabilloud T. Electrophoresis 2000 Oct;21(16):3329-44 Sample taken from the sodium carbonate extraction Santoni V, Kieffer S, Desclaux D, Masson F, Rabilloud T. Electrophoresis 2000 Oct;21(16):3329-44 Proteome analysis of the Pseudomonas aeruginosa, PAO1, Membrane proteome - Nouwens AS et al., Electrophoresis 2000 Nov;21(17):3797-809 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 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 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 Differences between normal and optimized sample solubilization 2 1 1. Outer membrane porin GRAVY –0,418 2. 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!! 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. Optimerization of IEF sample buffer 3 108 unsorted pig sperm cells Sodium carbonate extraction 7M Urea, 2M Thiourea, 1% ASB 14, 2mM TBP, 0,5% carrier ampholytes pH 4 solubilization 7M Urea, 2M Thiourea, 2% CHAPS, 2% SB 3-10, 1% ASB 14, 2mM TBP, 0,5% carrier ampholytes pH 7 pH 4 70 KDa 10 KDa pH 7 Optimization of the Na2CO3 extraction with respect to starting material 3 10 Marker proteins 8 – cells membrane associated Indicate cytoplasmic proteins ! 6 10 8 cells Cell sorting Pig sperm X and Y, Chromosomal difference: 3.5 % R1 Dead cells Sorted Pig sperm Pig Y Pig X 5 4 6 11 7 9 8 10 3 2 1 Marked spots differ in expression with > 1.7 22 Proteins showed expression changes > 1.7 - 11 of those could be identified by MALDI tandem mass spectrometry Spot number Protein identification Accession number Location Comments 1 Ras-related protein Rab-2A Q01971 Endoplasmic reticulum Lipid anchor 2 Ras-related protein Rab-2A Q01971 Endoplasmic reticulum Lipid anchor 3 Glutathione S-transferase, mu 5 P46439 Cytoplasmic ? 4 ATP synthase alpha chain P19483 Mitochondrial inner membrane 5 ATP synthase alpha chain P19483 Mitochondrial inner membrane 6 ATP synthase beta chain P00829 Mitochondrial 7 ATP synthase beta chain P00829 Mitochondrial 8 Glutathione S-transferase, mu 5 P46439 Cytoplasmic ? 9 Voltage-dependent anion-selective channel protein 2 P45880 Outer mitochondrial membrane 10 Voltage-dependent anion-selective channel protein 3 Q9Y277 Outer mitochondrial membrane 11 Outer dense fiber 2 protein O35496 Sperm tail membrane protein 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. Overview of the 2 D gel experiment KDa Total number of proteins detected on the gel. 300 Total number of protein spots tried identified from the 2 D gel. 118 Number of spot where the protein is identified. 52 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 90 30 3 pH 10 The majority of the proteins identified using the 2DE approach were membrane proteins derived from either the mitochondrial membrane or the sperm tail. Shaving membranes Whole Cell Limited proteolysis LC-MSMS Enriched Membrane sample LC-MSMS Christine C. Wu and John R. Yates, III. Nature biotechnology 21, 262-267 Liquid chromatography tandem mass spectrometry – membrane shaving Limited tryptic proteolysis of sorted cells 100 Reversed-phase capillary LC-MS 2 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 Time 80.00 90.00 100.00 110.00 Overviews of the LC-MS analysis MS-Tag Score Database Accession Protein Name Located to the surface 90.97 17388906 Cenexin 2 59.00 897763 Beta-tubulin 48.42 14194458 A-kinase anchor protein 3 47.79 5791527 Semenogelin I protein (SGI) 38.19 106185 GTP-binding protein Rab2 33.08 27713216 Tubulin alpha-3/alpha-7 chain 28.75 21493039 A kinase (PRKA) anchor protein 4 isoform 2 19.98 2253123 75 kDa fibrous sheath protein 17.87 497832 zona-pellucida-binding protein (sp38) Number of proteins identified using LC-MS/MS. 34 Number of known proteins identified by LC-MS/MS. 17 Number of hypothetical proteins identified by LC-MS/MS. 17 Number of identified known proteins found to be associated with the surface of the sperm cells. 10 (59%) Number of fragment spectra generated 1500 Number of ”peptide” fragment spectra. 497 16.59 20381082 LanC-like protein 1 16.30 27500209 Glypican-5 precursor 15.03 10305336 peroxiredoxin 5 14.86 8453169 Sperm-associated antigen 4 protein 14.83 1834491 EYA3 14.54 609541 Glucocorticoid-regulated endocrine protein 14.36 13507710 ropporin 14.33 4099365 glutathione-S-transferase, mu 5 The two techniques are highly complementary 2 DE Advantages • Separation of protein isoforms (modifications) • More reliable protein identifications! • High sequence coverage Disadvantages • Poor detection of low abundant proteins • Poor detection of basic proteins •Need more material than LC-MSMS • Quantitation can be relatively hard LC-MSMS Advantages • Sensitive (below pmol level) • Identification of ”difficult” proteins • Quantitation possible – e.g. ICAT/Stable isotope labeling 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. Overall the two techniques complements each other 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. 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. Pancreas ß-celle Muskelfiber Blodkar Insulin Glucose Copenhagen Model IL-1 ? Virus ? Chemicals ? Nutrition ? • • • • • • • • • • • • • IL-1 • • • • TNF a • • • Beta cells Klonal Tand Blymfocyt ekspansion Mø + NO • Nerup, J. Et al., 1994, Diabetologia 37 (Suppl 2), S82-S89. • O 2• • • • • • • • • IFN g IL-1 Mø / EC Th lymphocyte O 2- NO Cytokines => inducible nitric oxide synthase expression => NO generation Investigation of the effect of Interleukin-1 on isolated islets Islet isolation Cytokines => iNOS expression => NO generation => -cell destruction L-Arginine analogs (NMMA) S35-methionine IL-1ß + S35-methionine SDS PAGE Mass spectrometric protein identification H. U. Andersen et al, Electrophoresis 1997 P. Mose Larsen et al, Diabetes, 2001 pH 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 Plasma membrane sub-proteome - strategy SILAC: Stable isotop labeling with isotop amino acids Cell line in normal amino acid media Cell line in media containing Lys(13C) + Arg(13C,15N) IL - 1 + IL - 1 Reference Experiment 1 Pool 1:1 Cell lysis - mechanical in succrose buffer Trypsin digestion TiO2 purification Flowthrough Bound - phosphopeptides Succrose centrifugation and Na2CO3 treatment LC-MSMS Centrifugation Solubilization using SDS sample buffer SDS - PAGE LC-MSMS Tumor-associated calcium signal transducer 1 [Rattus norvegicus] Plasma membranes In-solution trypsin digestion EMGEIHR PM fraction SILAC, Trp, SCX, 10mM KCL u04963MRL 1654 (34.497) Sm (SG, 3x3.00); Cm (1652:1655) Strong cation exchange LC-MSMS 1: TOF MS ES+ 148 436.26 100 5 Da % 441.26 436.76 441.77 437.26 443.28 434.26 434.77 435.31 438.27 439.28 440.78 442.28 439.79 443.78 444.79 442.78 435.78 0 m/z 434 435 436 437 438 439 440 441 442 443 444 10 mM KCL fraction GESLFHSSK PM fraction SILAC, Trp, SCX, 10mM KCL u04963MRL 1681 (38.088) Cm (1679:1682) 1: TOF MS ES+ 135 496.30 100 3 Da 496.32 499.30 496.28 495.33 496.81 % 494.33 497.31 495.83 494.83 495.76 494.61 501.32 499.80 497.84 497.38 496.39 498.32 501.83 500.30 498.82 500.81 0 m/z 495 496 497 498 499 500 501 Cation-independent mannose-6-phosphate receptor LVSFHDDSDEDLLHI.- L+H C-terminal peptide from protein – can not be quantified by SILAC Golgi vesicular membrane trafficking protein p18 SLSIEIGHEVK L H Glycosyl-phosphatidylinositol anchored proteins: Membrane attached surface proteins GPI-anchored protein C terminus Man Ins Man P Acts as • enzymes • adhesion molecules • receptors • antigens •… P DAG Interior Man Widespread class of membrane proteins Plasma membrane Exterior GlcN Ethanolamine NH2 Ferguson et al. (1988) Science 239, 753-759 Strategy for isolation and identification of GPI-anchored proteins Cell culture Isolation of membrane fraction Isolation of GPI proteins •PI-PLC treatment •Two-phase separation PI-PLC SDS-PAGE of extracted protein LC-MSMS Identification of GPI-proteins by nanoLC-MS/MS Elortza, F et al, 2004 Identification of proteins in human lipid rafts 10 slices LC-MS/MS DB search 16 proteins Trypsin Literature Sequence analysis 6 GPI-anchored proteins Human GPI-proteins (lipid rafts) Accession no. • Alkaline phosphatase P05186 • Decay acceleration factor P08174 • Folate receptor 1 P15328 • CD59 glycoprotein P13987 • Carboxypeptidase P14284 • Urokinase plasminogen activator receptor Q03405 # peptides 11 9 8 3 2 1 OTHER: Fibronectin receptor-CD29 (P05556), Galactoprotein-CD49c (P26006), Melanoma adhesion molecule-CD146 (P431214), F2 heavy chain antigen-CD98 (P08195), EpicanCD44 (P16070), 78 kDa glucose-regulated protein (P11021), Mesotheline/megakaryocyte potentiation factor (Q9UK57), Actin /g actin (P02571/P02572), Voltage dependent anion channel (P21796), B-cell antigen receptor complex associated protein alpha-chain-CD79 (P11912). 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. Isolation of GPI-anchored proteins from A. thaliana cell line B A 100 PI-PLC Band # + - M kDa 116 1 2 3 4 5 6 7 8 9 10 11 12 % 0 32 36 40 97 66 44 48 Tim e (min) 52 C 100 45 MS 660.96 % 31 13 14 15 BPI 0 900 700 500 1100 D 21 R 16 D L S I E S D G D D V y4 100 y5 y1 14 m/z 1300 % y2 MS/MS y7 y9 y6 y3 y10 y8 y11 0 100 300 500 700 900 1100 1300 m/z Identification of GPI-anchored proteins in A. thaliana by LCMS/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 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……….. Conclusion Membrane proteins can be efficiently separated by 2 DE using 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. 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