Mass Spectrometric Quantitation of Proteins and Metabolites

Mass Spectrometric Quantitation of Proteins and Metabolites

Protein1: Structure &Interactions (Last week) Protein interaction codes(s)? Real world programming Pharmacogenomics : SNPs Chemical diversity : Nature/Chem/Design Target proteins : structural genomics Folding, molecular mechanics & docking Toxicity animal/clinical : cross-talk 1 Protein2: Properties & Quantitation Separation of proteins & peptides Protein localization & complexes Peptide identification (MS/MS) Database searching & sequencing. Protein quantitation

Absolute & relative Protein modifications & crosslinking Protein - metabolite quantitation 2 Why purify? Reduce one source of noise (in identification/quantitation) Prepare materials for in vitro experiments (sufficient causes) Discover biochemical properties 3 (Protein) Purification Methods Charge: ion-exchange chromatography, isoelectric focusing Size: dialysis, gel-filtration chromatography, gel-electrophoresis, sedimentation velocity

Solubility: salting out Hydrophobicity: Reverse phase chromatography Specific binding: affinity chromatography Complexes: Immune precipitation ( crosslinking) Density: sedimentation equilibrium 4 Protein Separation by Gel Electrophoresis Separated by mass: Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Sensitivity: 0.02ug protein with a silver stain. Resolution: 2% mass difference. Separated by isoelectric point (pI): polyampholytes pH gradient gel. Resolution: 0.01 pI. 5

Comparison of predicted with observed protein properties (localization, postsynthetic modifications) E.coli Link et al. 1997 Electrophoresis 18:1259-313 (Pub) 6 Computationally checking proteomic data Property

Basis of calculation Protein charge Protein mass Peptide mass Peptide LC Subcellular Expression RKHYCDE (N,C), pKa, pH (Pub) Calibrate with knowns (complexes) Isotope sum (incl.modifications) aa composition linear regression Hydrophobicity, motifs (Pub) Codon Adaptation Index (CAI) 7 Protein2: Today's story & goals Separation of proteins & peptides

Protein localization & complexes Peptide identification (MS/MS) Database searching & sequencing. Protein quantitation Absolute & relative Protein modifications & crosslinking Protein - metabolite quantitation 8 Cell fraction: Periplasm 2D gel: SDS mobility isoelectic pH Mr Link et al. 1997 Electrophoresis

18:1259-313 (Pub) 9 Cell localization predictions TargetP: using N-terminal sequence discriminates mitochondrion, chloroplast, secretion, & "other" localizations with a success rate of 85%. (pub) Gromiha 1999, Protein Eng 12:557-61. A simple method for predicting transmembrane alpha helices with better accuracy. (pub) Using the information from the topology of 70 membrane proteins... correctly identifies 295 transmembrane helical segments in 70 membrane proteins with only two overpredictions. 10 Isotope calculations Mass resolution 0.1% vs. Symbol

-----H(1) C(12) N(14) O(16) S(32) Mass ---------1.007825 12.000000 14.003074 15.994915 31.972072 1 ppm Abund. -----99.99 98.90 99.63

99.76 95.02 Symbol -----H(2) C(13) N(15) O(17) S(33) Mass ----------2.014102 13.003355 15.000109 16.999131 32.971459 Abund. ------0.015

1.10 0.37 0.038 0.75 11 Computationally checking proteomic data Property Basis of calculation Protein charge Protein mass Peptide mass Peptide LC Subcellular Expression

RKHYCDE (N,C), pKa, pH (Pub) Calibrate with knowns (complexes) Isotope sum (incl.modifications) aa composition linear regression Hydrophobicity, motifs (Pub) Codon Adaptation Index (CAI) 12 trypsin High Performance Liquid Chromatography 13 Mobile Phase of HPLC

The interaction between the mobile phase and sample determine the migration speed. Isocratic elution: constant migration speed in the column. Gradient elution: gradient migration speed in the column. 14 Stationary Phase of HPLC The degree of interaction with samples determines the migration speed.

Liquid-Solid: polarity. Liquid-Liquid: polarity. Size-Exclusion: porous beads. Normal Phase: hydrophilicity and lipophilicity. Reverse Phase: hydrophilicity and lipophilicity. Ion Exchange. Affinity: specific affinity. 15 Sereda, T. et al. Effect of the -amino group on peptide retention behaviour in reversed-phase chromatography. (The calculated curve is displaced upward for clarity) Wilce, et al. High-performance liquid chromatography of amino acids, peptides and

proteins. Journal of Chromatography, 632 (1993) 11-18. RP-LC calculated observed Empirical linear regression varies with type of LC-material -NH3+? C18 no yes no W 10.1 9.3 9.8 F 8.8 5.5 8.8 L 7.5 4.6 9.5

I 5.8 3.0 8.4 M 4.8 3.0 2.6 Y 4.5 3.1 6.1 V 3.5 1.3 4.9 C 3.4 2.9 0.5 P 2.7 0.7 2.8 E 0.3 0.5 0.8 A 0.2 0.1 1.7 D 0.0 0.6 1.1 G 0.0 0.0 0.4 T -0.1 1.0 1.8 S -0.8 -0.1 0.3 Q -0.9 0.0 -0.7 N -3.0 -2.1 0.0 R -3.1 -2.1 2.4 H -3.3 -1.5 0.6 K -3.5 -1.6 0.0 16

A Map is Like a 2D Peptide Gel First Dimension: Reverse Phase Chromatography Separation By Hydrophobicity R A P S V MW C A G F L E K

Q G RT min C C T K D m/z Second Dimension: Mass Spectrometry Separation by Mass

17 What Information Can Be Extracted From A Single Peptide Peak abundance Isotopic Variants of DAFLGSFLYEYSR rt m/z abundance @ 36.418 min 0 X 13C 1 X 13C 2 X 13C

3 X 13C K.Leptos 2001 18 m/z Directed Analysis of Large Protein Complexes by 2D separation: strong cation exchange and reversed-phased liquid chromatography. Link, et al. 1999, Nature Biotech. 17:676-82. (Pub) 19 A new

40S subunit protein >9 5-9 2-4 1 #peptides #uniquely identified / #genes 1/1 2/2 1/2 0/2 20

Protein2: Properties & Quantitation Separation of proteins & peptides Protein localization & complexes Peptide identification (MS/MS) Database searching & sequencing. Protein quantitation Absolute & relative Protein modifications & crosslinking Protein - metabolite quantitation 21 The Finnigan LCQ: An ESI-QIT Mass Spectrometer Electro-Spray Ionization chamber

Mass Analyzer/Detector 22 Tandem Mass Spectrometry Quadrople Q1 scans or selects m/z. Q2 transmits those ions through collision gas (Ar). Q3 Analyzes the resulting fragment ions. Siuzdak, Gary. The emergence of mass spectrometry in biochemical research. Proc. Natl. Acad. Sci. 1994, 91, 11290-11297. Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1994, 11, 601. 23

Ions 24 Peptide Fragmentation and Ionization 25 Tandem Mass Spectra Analysis y b 26 Gygi et al. Mol. Cell Bio. (1999) Mass Spectrum Interpretation

Challenge It is unknown whether an ion is a b-ion or an y-ion or else. Some ions are missing. Each ion has multiple of isotopic forms. Other ions (a or z) may appear. Some ions may lose a water or an ammonia. Noise. Amino acid modifications. 27 A dynamic programming approach to de novo peptide sequencing via tandem mass spectrometry Chen et al 2000. 11th Annual ACM-SIAM Symp. of Discrete Algorithms pp. 389-398. 28

SEQUEST: Sequence-Spectrum Correlation Given a raw tandem mass spectrum and a protein sequence database. For every protein in the database, For every subsequence of this protein Construct a hypothetical tandem mass spectrum Overlap two spectra and compute the correlation coefficient (CC). Report the proteins in the order of CC score. Eng, et al. 1994, Amer. Soc. for Mass Spect. 5: 976-989 (Sequest) 29 Protein2: Properties & Quantitation

Separation of proteins & peptides Protein localization & complexes Peptide identification (MS/MS) Database searching & sequencing. Protein quantitation Absolute & relative Protein modifications & crosslinking Protein - metabolite quantitation 30 Expression quantitation methods RNA Protein Genes immobilized labeled RNA RNAs immobilized labeled genesNorthern gel blot

QRT-PCR Reporter constructs Fluorescent In Situ (Hybridization) Tag counting (SAGE) Differential display Antibody arrays Westerns -nonesame same (Antibodies) -nonemass spec 31 Molecules per cell E.coli/yeast Human Individual mRNAs:

10-1 to 103 10-4 to 105 Proteins: 10 to 106 10-1 to 108 32 MS Protein quantitation R=.84 Yeast Protein ESI-MS Quantitation 10000 Day 2 Measure 1000 y = 0.8754x + 0.1573 R2 = 0.8381

100 10 1 0.1 0.1 Link, et al 1 10 100 1000

Day 1 measure 33 MS quantitation reproducibility Sample: Angiotensin, Neurotensin, Bradykinin Map: 600 700 m/z Coefficients of Variance CV = 5 Frequency 4 3 2 1

0 CV 34 Correlation between protein and mRNA abundance in yeast Gygi et al. 1999, Mol. Cell Biol. 19:1720-30 (Pub) 35 Normality tests See Weiss 5th ed. Page 920. Types of non-normality: kurtosis, skewness (www) (log) transformations to normal.

Futcher et al 1999, A sampling of the yeast proteome. Mol.Cell.Biol. 19:7357-7368. (Pub) 36 Spearman correlation rank test rs = 1 - {6S/(n3-n)} Rank (from 1 to n, where n is the number of pairs of data) the numbers in each column. If there are ties within a column , then assign all the measurements that tie the same median rank. Note, avoids ties (which reduce the power of the test) by measuring with as fine a scale as possible. S= sum of the square differences in rank. (ref) X Y Rx Ry 1 8 1 4 6 2 3 1 6 3 3 2 n=4 6 4 3 3 37

Correlation of (phosphorimager 35 S met) protein & mRNA rp = 0.76 for log(adjusted RNA) to log(protein) rs = .74 overall; 0.62 for the top 33 proteins & 0.56 (not significantly different) for the bottom 33 proteins 38 Observed (Phosphorimage) protein levels vs. Codon Adaptation Index (CAI) Codon Adaptation Index (CAI) Sharp and Li (1987); fi

is the relative frequency of codon i in the coding sequence, and Wi the ratio of the frequency of codon i to the frequency of the major codon for the same amino-acid. ln(CAI)= fi ln (Wi) i=1,61 39 ICAT Strategy for Quantifying Differential Protein Expression. X= H or D

Gygi et al. Nature Biotechnology 40 (1999) Mass Spectrum and Reconstructed Ion Chromatograms. Gygi et al. Nature Biotechnology 41 (1999) Protein & mRNA Ratios +/- Galactose Ideker et al 422001 Protein2: Properties & Quantitation Separation of proteins & peptides Protein localization & complexes

Peptide identification (MS/MS) Database searching & sequencing. Protein quantitation Absolute & relative Protein modifications & crosslinking Protein - metabolite quantitation 43 Post-synthetic modifications Radioisotopic labeling: PO4 S,T,Y,H Affinity selection: Cys: ICAT biotin-avidin selection PO4: immobilized metal Ga(III) affinity chromatography(IMAC) Specific PO4 Antibodies Lectins for carbohydrates

Mass spectrometry 44 32P labeled phoshoproteomics Low abundance cell cycle proteins not detected above background from abundant proteins Futcher et al 1999, A sampling of the yeast proteome. Mol.Cell.Biol. 19:7357-7368. (Pub) 45 Natural crosslinks Disulfides Collagen Ubiquitin

Fibrin Glycation Adeno primer proteins Cys-Cys Lys-Lys C-term-Lys Gln-Lys Glucose-Lys dCMP-Ser 46 Crosslinked peptide Matrixassisted laser desorption ionization PostSource Decay (MALDI-PSDMS) tryptic digest of BS3 cross-linked

FGF-2. Cross-linked peptides are identified by using the program ASAP and are denoted with an asterisk (9). (B) MALDI-PSD spectrum of cross-linked peptide E45R60 (M + H+ = m/z 2059.08). 47 Constraints for homology modeling based on MS crosslinking distances The 15 nonlocal throughspace distance constraints generated by the chemical crosslinks (yellow dashed lines) superimposed on

the average NMR structure of FGF-2 (1BLA). The 14 lysines of FGF-2 are shown in red. Young et al 2000, PNAS 97: 5802 (Pub) 48 Homology modeling accuracy 100 90 80 70 % sequence identity

60 50 40 30 20 1 1.5 2 2.5 Swiss-model RMSD of the test set in Angstroms 3 3.5

4 49 Top 20 threading models for FGF ranked by crosslinking constraint error 50 Protein2: Properties & Quantitation Separation of proteins & peptides Protein localization & complexes Peptide identification (MS/MS) Database searching & sequencing. Protein quantitation Absolute & relative Protein modifications & crosslinking

Protein - metabolite quantitation 51 Challenges for accurately measuring metabolites Rapid kinetics Rapid changes during isolation Idiosyncratic detection methods: enzyme-linked, GC, LC, NMR (albeit fewer molecular types than RNA& protein) 52 1634 Metabolite Masses 256 amino acids 600 Y=

400 Frequency 598 have identical mass e.g. Ile & Leu = 131.17 200 0 Databases 160 240 80 320 560 800 1040 1280 1520 X = Mass

Karp et al. (1998) NAR 26:50. EcoCyc; Selkov, et al. (1997) NAR 25:37. WIT Ogata et al. (1998) Biosystems 47:119-128 KEGG 53 Y= RP LC retention time in min. I L (higher hydrophocity)

W X = Mass 54 Wunschel J Chromatogr A 1997, 776:205-19 Quantitative analysis of neutral & acidic sugars in whole bacterial cell hydrolysates using highperformance anion-exchange LC-ESI-MS2. (Pub)

Metabolite fragmentation & stable isotope labeling 55 Isotopomers Klapa et al. Biotechnol Bioeng 1999; 62:375. Metabolite and isotopomer balancing in the analysis of metabolic cycles: I. Theory. (Pub) "accounting for the contribution of all pathways to label distribution is required, especially ... multiple turns of metabolic 56 cycles... 13C (or 14C) labeled substrates." MetaFoR: Metabolic Flux Ratios Fractional 13C labeling > Quantitative 2D NMR Why use amino acids from proteins rather than metabolites directly?

Sauer J et al. Bacteriol 1999;181:6679-88 (Pub) Szyperski et al 1999 Metab. Eng. 1:189. Dauner et al. 2001 Biotec Bioeng 76:144 57 A functional genomics strategy that uses metabolome data to reveal the phenotype of silent mutations -40C MeOH> 80C EtOH > Cobas Enzymatic BioAutoanalyser & Quantitative 1H NMR 0 to 4.4 ppm (1300 measures) Raamsdonk et al. 2001 Nature Biotech 19:45. 58 Types of interaction models

Quantum Electrodynamics Quantum mechanics Molecular mechanics Master equations subatomic electron clouds spherical atoms (101Pro1) stochastic single molecules (Net1) Phenomenological rates ODE Flux Balance Thermodynamic models Concentration & time (C,t) dCik/dt optima steady state (Net1) dCik/dt = 0 k reversible reactions

Steady State Metabolic Control Analysis Spatially inhomogenous models dCik/dt = 0 (sum k reactions) d(dCik/dt)/dCj (i = chem.species) dCi/dx Increasing scope, decreasing resolution 59 How do enzymes & substrates formally differ? E A EA ATP

E EATP EB B E2+P ADP EP 60 Catalysts increase the rate (&specificity) without being consumed. Enzyme rate equations with one Substrate & one Product

E S P dP/dt = V (S/Ks - P/Kp) 1 + S/Ks + P/Kp S As P approaches 0: dP/dt = V 1+ Ks/S 61 Enzyme Kinetic Expressions Phosphofructokinase

vPFK F 6 P PFK K F 6P 1 F 6 P K PFK F 6P PFK vmx N PFK PFK 0

N PFK 1 L Mg ATP PFK K Mg ATP 1 Mg ATP PFK K Mg ATP ATPfree 1 PFK K ATP 4

4 Mg 1 PFK K Mg 4

4 AMP F 6 P PFK 1 PFK 1 K AMP K F 6P Allosteric kinetic parameters for AMP, etc. 62 Human Red Blood Cell

ODE model ADP ATP ADP ATP 1,3 DPG NADH NAD 3PG GA3P 2,3 DPG FDP

2PG DHAP PEP F6P ADP ATP R5P GA3P F6P PYR G6P GLCe GL6P GO6P

ADP ATP NADP NADP NADPH NADPH ADP 2 GSH GLCi K+ ATP ADP

Na+ GSSG ODE model INOe X5P S7P GA3P E4P F6P NADPH NADP ADO ADE

ADP INO IMP ATP LACe Cl- HCO3- PRPP AMP PRPP HYPX LACi

pH AMP ATP ADOe NADH NAD RU5P ATP R1P R5P

ADEe Jamshidi et al. 2000 (Pub) 63 Red Blood Cell in Mathematica ODE model Jamshidi et al. 2000 (Pub) 64 Protein2: Properties & Quantitation Separation of proteins & peptides Protein localization & complexes

Peptide identification (MS/MS) Database searching & sequencing. Protein quantitation Absolute & relative Protein modifications & crosslinking Protein - metabolite quantitation 65

Recently Viewed Presentations

  • Transistors, Logic Gates and Karnaugh Maps

    Transistors, Logic Gates and Karnaugh Maps

    All inputs are filled in with light blue. In this version, more inputs differing by one bit only are in adjacent positions. * Karnaugh-map This way of arranging truth tables combined with the rules for simplifying Boolean expressions goes by...
  • Can Drug-Coated Balloons Be a Solution for Complex Peripheral ...

    Can Drug-Coated Balloons Be a Solution for Complex Peripheral ...

    Total IN.PACTPooled Analysis Initiative (cont) Total IN.PACT Pooled Analysis Initiative - Primary Endpoints. All SubjectsBaseline Lesion/Procedural Characteristics. Primary Efficacy Outcome. Total IN.PACTImaging (Patency) Analysis.
  • Supporting K-6 Students with the Online Reference Centre

    Supporting K-6 Students with the Online Reference Centre

    Embedded science experiments, projects, and educational games related to the content of the database. Aligns with . 95%. ... Pebble GO Social Studies. Culturegrams. The Canadian Encyclopedia. ... Supporting K-6 Students with the Online Reference Centre
  • RISE OF THE NAZI PARTY AND NAZI IDEOLOGY

    RISE OF THE NAZI PARTY AND NAZI IDEOLOGY

    World War I. Austria - moved to Germany - avoid evasion arrest . Germany - enlisted in 1914. served as a dispatch runner. top rank - corporal
  • Module 4: Twin Transformations Creation Care as a

    Module 4: Twin Transformations Creation Care as a

    MORAL OBLIGATIONS "If ever there were an urgent moral and spiritual issue, this is it.We risk a two-fold betrayal - of our responsibility to the Creator for the good stewardship of his creation, and of our responsibility to our vulnerable...
  • Abdominal wall and Retroperitoneum

    Abdominal wall and Retroperitoneum

    Omphaloceles occur in approximately 1:4,000 live births, and include a spectrum of midline defects that range from large (usually containing liver and bowel) to small (which may contain only 1 or 2 bowel loops). The exteriorized viscera are contained by...
  • 1999 Corporate Tax Update

    1999 Corporate Tax Update

    Global Intangible Low-taxed Income (GILTI) applies to Controlled Foreign Corporation (CFC) for tax years after 12-31-17. US shareholder who owns 10% or more of the CFC must include their share of GILTI as current taxable income, whether distributed or not
  • Institutional Repositories - University of Calgary

    Institutional Repositories - University of Calgary

    Institutional Repositories Preserving Digital Scholarship Mark Jordan, Simon Fraser University Kathleen Shearer, Canadian Association of Research Libraries Here Today, Gone Tomorrow Winnipeg, MB, June 2, 2004