DAH3.1 Mass Spectrometry Definition of the Proteome Why bother studying it

DAH3.1 Mass Spectrometry 		Definition of the Proteome Why bother studying it www.phwiki.com

DAH3.1 Mass Spectrometry Definition of the Proteome Why bother studying it

Galloway, Roger, News Director has reference to this Academic Journal, PHwiki organized this Journal DAH3.1 Mass Spectrometry Kathryn Lilley Cambridge Centre as long as Proteomics Department of Biochemistry University of Cambridge k.s.lilley@bioc.cam.ac.uk www.bio.cam.ac.uk/proteomics/ Part III Systems Biology Definition of the Proteome The analysis of the entire PROTEin complement expressed by a genOME. Wasinger et al Electrophoresis 16 (1995) Could be: Cellular extract Secreted fluid Tissue Whole organism Why bother studying it Cambridge Centre as long as Proteomics A Proteomicist’s Tools Mass spectrometry Protein in addition to peptide separation methods Databases in addition to software Validation tools Western blotting GFP tagging in addition to confocal microscopy

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Instruments as long as mass analysis Mass Spectrometers measure m/z of gaseous ion Mass spectrometers comprise: A source which is responsible as long as ionising the sample, e.g. electrospray, laser desorption An analyser which separates in addition to 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 Outline Proteomics workflows Protein identification Post translational modification Protein analysis on pure proteins/complexes Mass of protein Modification status can be difficult to deconvolute with many iso as long as ms Higher order structures ability to spray whole complexes in addition to look at components in addition to stoichiometries Low through put methods Usually carried out on pure proteins or complexes Already know what the protein is

Protein analysis on complex mixtures For more complex samples you cannot purify each one in addition to 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 as long as the protein 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 Which proteins are present Trypsin Peptides 1D gel 2D gel Solution Digest

Workflow 1 MALDI/MS Peptide mass fingerprinting Excise Digest Apply to MALDI ToF Matrix Assisted Laser Desorption Ionisation (MALDI) a-cyano-4-hydroxycinnamic acid 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+ ) MALDI Tof MS

Trypsin K R R K 1457.35 1765.33 1975.72 2055/78 2589.31 Matrix assisted desorption time of flight mass spectrometry Mass list Database search of virtual trypsin digested translated genome Identification !!!! Peptides Peptide Mass Fingerprinting Score = 110 Limitations Only works well as long as purified proteins Require well annotated genome Strengths Quick Cheap

Workflow 2 HPLC peptide separation Electrospray ionisation LC MS/MS Digest Chromatography separations High Per as long as mance 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 Bind peptides Elute with gradient e.g. acetonitrile as long as reverse phase Increasing salt as long as SCX Mass Spectrometry Western blotting GFP tagging Immuno- histochemistry Enzyme assay 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

T in addition to em Mass Spectrometry LC-MS/MS: Data Dependent Acquisition in MS Q CID ToF Precursor ion selection based on intensity Precursors scanned out of first quad. Collision induced dissociation All fragment ions analysed Typical output List of peptide masses Precursor mass (parent ion mass) Fragment ion masses y-ions b-ions Protein identification Search engines MASCOT – http://www.matrixscience.com SEQUEST – fields.scripps.edu/sequest/ X ! T in addition to em -www.thegpm.org/t in addition to em/index.html Phenyx- www.genebio.com/products/phenyx/

Score = 960 MUDPIT Data dependent acquisition means that only the most intense ions at any given time are taken as long as MS/MS To improve coverage, peptide simplification is required MUDPIT Multi dimensional protein Identification technology Washburn et al (2001) Nat. Biotech 19:242 Strengths in addition to 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

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Can you be sure Validation GFP Using molecular biology techniques fuse gene encoding a fluorescent protein to your protein of interest. Western blotting Proteins from gel blotted onto PVDF membrane Primary antibody – raised against your protein of interest Secondary antibody – raised against the first antibody constant regions Enzyme, fluorescent tag Quantitative Western Blotting on a System-wide Scale Quantitative western blotting of 75% of yeast proteome Ghaemmaghami et al, 2003 A massive amount of work Not transferable to many organisms GFP tagging of yeast proteome Huh et al, 2003 GFP tagged proteins 75% of the yeast proteome classified to 22 distinct location

Systems wide immuno-histochemistry Barbe et al, 2008 Antibodies to 488 proteins applied to 3 different human cell lines in addition to images stored in addition to publically accessible Blue = DAPI staining of nucleus 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 Protein Iso as long as m Analysis Proteins may be: Covalently modified Truncated Dimerised

Truncations Easiest way look at peptide coverage N-terminal peptide analysis Edman degradation COFRADIC Combined fractional diagonal chromatography Acetylate total proteins with acetic anhydride. All amino groups acetylated Digest with trypsin Carry out liquid chromatography (usually reverse phase) in addition to collect peptides in fractions Modify all new N-termini generated with trypsin with TNBS (this makes the peptide very hydrophobic). Rerun all fractions using same LC conditions as be as long as e. Peptide which eluted in the same place are the original N-terminus, those that move are internal peptides. Ac Ac Ac Gevaert et al (2003) Nat. Biotech 21:566 This week Tuesday: 1pm Seminar by Matthias Mann 2.15pm Q in addition to A session with Prof. Mann in addition to lecture as long as me Wednesday: 9am – Practical Class 4pm Lecture from me

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