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DAH3.1 Mass Spectrometry

DAH3.1 Mass Spectrometry . Kathryn Lilley Cambridge Centre for Proteomics Department of Biochemistry University of Cambridge k.s.lilley@bioc.cam.ac.uk www.bio.cam.ac.uk/proteomics/. Part III Systems Biology. Cambridge Centre for Proteomics. Definition of the Proteome

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DAH3.1 Mass Spectrometry

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  1. DAH3.1 Mass Spectrometry Kathryn Lilley Cambridge Centre for Proteomics Department of Biochemistry University of Cambridge k.s.lilley@bioc.cam.ac.uk www.bio.cam.ac.uk/proteomics/ Part III Systems Biology

  2. Cambridge Centre for Proteomics Definition of the Proteome The analysis of the entire PROTEin complement expressed by a genOME. Wasinger et alElectrophoresis 16 (1995) Why bother studying it???? Could be: Cellular extract Secreted fluid Tissue Whole organism

  3. A Proteomicist’s Tools • Mass spectrometry • Protein and peptide separation methods • Databases and software • Validation tools • Western blotting • GFP tagging and confocal microscopy

  4. Instruments for mass analysis Mass Spectrometers measure m/z of gaseous ion Mass spectrometers comprise: A source which is responsible for ionising the sample, e.g. electrospray, laser desorption An analyser which separates and carries the ions to the detector e.g. Quadrupole, Ion-trap (mass range 2000-4000 m/z) TOF (time of flight) (mass range 0-200,000+ m/z) A detector e.g. electron multiplier

  5. Outline • Proteomics workflows • Protein identification • Post translational modification

  6. Protein analysis on pure proteins/complexes Mass of protein Modification status can be difficult to deconvolute with many isoforms Higher order structures ability to spray whole complexes and look at components and stoichiometries Low through put methods Usually carried out on pure proteins or complexes Already know what the protein is

  7. Protein analysis on complex mixtures For more complex samples you cannot purify each one and then analyse it. Methods need to be applied where proteins can be analysed simultaneously Proteins can be separated then analysed or converted to peptides which are then analysed The peptides act as surrogates for the protein

  8. Abundance Isoform status Quantitative Mass Spec Western blotting GFP tagging Immuno- histochemistry Enzyme assay Arrays Mass Spectrometry Western blotting Functional arrays Types of protein analysis Proteins present PPI SCL Function Mass Spectrometry Western blotting GFP tagging Immuno- histochemistry Enzyme assay Arrays Y2H Tagging + Mass Spectrometry Western blotting Structural studies Protein Arrays Biophysical assays (e.g.ITC, AUC) Mass Spectrometry GFP tagging Immuno- histochemistry Enzyme assay Functional arrays Enzyme assay Genetic approaches

  9. Mass Spectrometry Western blotting GFP tagging Immuno- histochemistry Enzyme assay Which proteins are present 1D gel 2D gel Solution Digest Trypsin Peptides

  10. Mass Spectrometry Western blotting GFP tagging Immuno- histochemistry Enzyme assay Workflow 1 • MALDI/MS • Peptide mass fingerprinting Excise Digest Apply to MALDI ToF

  11. Matrix Assisted Laser Desorption Ionisation (MALDI) a-cyano-4-hydroxycinnamic acid

  12. Reflectron Detector Matrix Suppression Lens Ion Beam Reflectron Assembly Gas Cell Sample target N2 Laser Linear Detector MALDI Tof MS The chemical matrix absorbs energy from the laser pulse which is transferred to the protein The sample ions are then accelerated towards the detector Principally produces M+H+ ions (sometimes M+2H+ )

  13. Peptide Mass Fingerprinting K Peptides R R K Trypsin Matrix assisted desorption time of flight mass spectrometry Identification !!!! 1457.35 1765.33 1975.72 2055/78 2589.31 Mass list Database search of virtual trypsin digested translated genome

  14. Score = 110

  15. Limitations Strengths Quick Cheap Only works well for purified proteins Require well annotated genome

  16. Mass Spectrometry Western blotting GFP tagging Immuno- histochemistry Enzyme assay MS CID MS Workflow 2 • HPLC peptide separation • Electrospray ionisation • LC MS/MS Digest

  17. Chromatography separations Bind peptides High Performance Liquid Chromatography (HPLC) Strong cation exchange (SCX) Separation based on net charge of peptide Weak anion exchange (WAX) Separation based on net charge of peptide Reverse phase (RP) Separation based on hydrophobicity Hydrophobic interaction chromatography (HILIC) Separation based on hydrophobicity Mass Spectrometry Western blotting GFP tagging Immuno- histochemistry Enzyme assay Elute with gradient e.g. acetonitrile for reverse phase Increasing salt for SCX

  18. LC-MS/MS Molecular ion (precursor) is accelerated into collision cell where it collides with an inert gas Some of the kinetic energy is converted to internal (vibrational) energy Peptide cleavage takes place largely at the peptide bond nearest a mobile proton Net result is: Detect positively charged fragments which contain either the original N-terminus or C-terminus of the peptide

  19. Tandem Mass Spectrometry LC-MS/MS:Data Dependent Acquisition in MS Q CID ToF Collision induced dissociation Precursor ion selection based on intensity Precursors scanned out of first quad. All fragment ions analysed

  20. Typical output • List of peptide masses • Precursor mass (parent ion mass) • Fragment ion masses • y-ions • b-ions

  21. Protein identification • Search engines MASCOT - http://www.matrixscience.com SEQUEST - fields.scripps.edu/sequest/ X ! Tandem -www.thegpm.org/tandem/index.html Phenyx- www.genebio.com/products/phenyx/

  22. Score = 960

  23. MUDPIT • Data dependent acquisition means that only the most intense ions at any given time are taken for MS/MS • To improve coverage, peptide simplification is required MUDPIT Multi dimensional protein Identification technology Washburn et al (2001) Nat. Biotech 19:242

  24. Strengths and weaknesses • Can be used with very complex mixtures of proteins • If the genome is not sequenced then sequence returned may show similarity or identity to related organisms • De novo sequencing • More time consuming • Equipment more expensive

  25. Can you be sure?Validation?? • GFP Using molecular biology techniques fuse gene encoding a fluorescent protein to your protein of interest. • Western blotting Enzyme, fluorescent tag Secondary antibody – raised against the first antibody constant regions Primary antibody – raised against your protein of interest Proteins from gel blotted onto PVDF membrane

  26. Quantitative Western Blotting on a System-wide Scale Quantitative western blotting of 75% of yeast proteome A massive amount of work Not transferable to many organisms Ghaemmaghami et al, 2003

  27. GFP tagging of yeast proteome GFP tagged proteins 75% of the yeast proteome classified to 22 distinct location Huh et al, 2003

  28. Systems wide immuno-histochemistry Antibodies to 488 proteins applied to 3 different human cell lines and images stored and publically accessible Blue = DAPI staining of nucleus Barbe et al, 2008

  29. Abundance Isoform status Quantitative Mass Spec Western blotting GFP tagging Immuno- histochemistry Enzyme assay Arrays Mass Spectrometry Western blotting Functional arrays Types of protein analysis Proteins present PPI SCL Function Mass Spectrometry Western blotting GFP tagging Immuno- histochemistry Enzyme assay Arrays Y2H Tagging + Mass Spectrometry Western blotting Structural studies Protein Arrays Biophysical assays (e.g.ITC, AUC) Mass Spectrometry GFP tagging Immuno- histochemistry Enzyme assay Functional arrays Enzyme assay Genetic approaches

  30. Protein Isoform Analysis Isoform status Proteins may be: • Covalently modified • Truncated • Dimerised Mass Spectrometry Western blotting Functional arrays

  31. Post Translational Modifications Isoform status 100s of different PTMs Most commonly characterised • Phosphorylation xxx • Acetylation/Methylation x • Ubiquitination x • Sumoylation xx • Glycosylation • S-nitrosylation xxxx • ……………………. Mass Spectrometry Western blotting Functional arrays

  32. Phosphorylation Phosphorylation is a very important PTM Signalling pathways Protein conformational changes Serine, threonine and tyrosine are the most frequently phosphorylated residues

  33. PhosphorylationMost popular approaches • 32P incorporation to track peptides and quantify recovery • Isolate / enrich phosphopeptides by metal-chelation chromatography • Use triple-quad and hybrid-Tof instruments to look for neutral mass loss • Prediction algorithms

  34. Problems with Phosphoproteomics • Phospho groups are highly dynamic • Phospho tyrosine is very rare • Phosphopeptides ionise poorly, they tend to be very acidic • The phosphate group tends to fall off pSer and pThr during MS/MS

  35. Phospho-protein and -peptide enrichment • Phospho-tyrosine • good antibodies • Phospho-serine and phospho-threonine • Metal chelate chromatography • Ion exchange chromatography

  36. COOH OH P O O O Fe3+ NTA NH2 Agarose Bead Enrichment methods for phosphopeptides Immobilized metal affinity chromatography Titanium (IMAC) Dioxide Ferric or Gallium columns most usually employed

  37. Mass Spectrometry of Phosphopeptides • Standard methods • Neutral loss • ETD

  38. Neutral Loss RLSIELTNSLLR RLSIELTNSLLR P P Precursor ion Loss of PO3- group Intense fragment ion peak at m/z = 747.94 (2+) m = 98 Da, z = 2+ m/z = 698.94 (747.94 – 49) m/z = 49

  39. Electron Transfer Dissociation (ETD). • Gentler fragmentation than CID • Preserves post-translational modifications, such as phosphorylation • Produces c and z ions • Better sequence coverage than CID

  40. [M + 3H]3+ + A- [M + 3H]2+• + A Electron Transfer Dissociation (ETD) [M + 3H]2+• [C+2H]1+ + [Z+H]1+• C Z Fluoranthene Radical Anion (Good Electron Donor/ETD Reagent) R >1 eV Electron “Thermal” - e- + R

  41. CID:Prominent loss of phosphate (M+3H-H3PO4)3+ Parent ion = 571.22 538.25 = loss of phosphoric acid 538.25 Cambridge_A1 Cambridge_A1 Cambridge_A1 Cambridge_A1 T: T: 100 100 100 95 95 95 90 90 90 85 85 85 80 80 80 KSLSSNVGSTVKPPTK 75 75 75 70 70 70 65 65 65 60 55 Relative Abundance 50 45 40 35 30 25 20 15 559.02 10 742.61 5 0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z

  42. ETD:KSLSSNVGSTVKPPTK 856.18 100 95 90 85 80 75 70 65 60 1711.71 571.03 55 Relative Abundance 50 45 40 35 30 25 791.14 1694.83 20 754.38 15 1581.70 957.40 600.31 1479.61 1112.54 10 870.42 554.32 714.42 1157.58 233.32 1058.49 5 1399.72 513.29 653.44 1286.90 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

  43. Methylation/Acetylation More straightforward, but will be issues with digestion rates if trypsin is used Can enrich, antibody affinity capture to acetyl-lysine Pang et al (2010) Identification of arginine- and lysine-methylation in the proteome of Saccharomyces cerevisiae and its functional implications BMC Genomics 2010, 11:92 Choudhary, et al. (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions, Science325, 834-840.

  44. Ubiquitin Ubiquitin is a small highly conserved eucaryote protein, it attaches to other protein via lysine residues by ubiqutin ligases, often marking proteins for degradation by the proteasome system MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG Tryptic digestion UBIQUITIN ..R-L-R-G-G- ………….X-X-X-K-X-X………………. SUBSTRATE PROTEIN Kirkpatrick D.S., Denison C., and Gygi S.P., Weighing in on ubiquitin: the expanding role of mass-spectrometry-based proteomics. Nat Cell Biol, 2005. 7(8): p. 750-7

  45. R G D E L Q K G A F LI G G

  46. SUMO Small Ubiquitin-like Modifier 3 (4) versions Many functions including stability, nuclear-cytosolic transport, and transcriptional regulation Sadly there is no well placed tryptic site or site for any other common protease near the point at which it attaches to its modification target MSMS spectra are thus a mess, as two sets of –b and –y ions will be produced per with SUMO modification Automated identification of SUMOylation sites using mass spectrometry and SUMmOn pattern recognition software. Pedrioli PG, et al. Nat Methods. 2006 3(7):533-9.

  47. Galisson et al (2011) Mol. Cell Prot. A novel proteomics approach to identify SUMOylated proteins and their modification sites in human cells Engineered SUMO is Hek293 cells to have strategically located tryptic site and (His)6 for purification

  48. Bruderer et al EMBO Rep. 2011 Feb;12(2):142-8..Purification and identification of endogenous polySUMO conjugates. E3 ligase inactive RNF4 fragment binds polySUMO

  49. Glycosylation Common, up to 50% of human proteins are glycosylated Need to fond site of attached, using N-linked (Asn) or O-linked (Ser) Also need to determine structure of glycosyl group Very complex, highly combinatorial The most challenging PTM for high throughput proteomics Enrichment possible with lectin affinity chromatography

  50. Typical schema used in large scale glycoproteome analysis

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