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Protein1: Last week's take home lessons
• 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
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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
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Why purify?
• Reduce one source of noise (in identification/quantitation)• Prepare materials for in vitro experiments (sufficient causes)• Discover biochemical properties
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(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
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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.
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Link et al. 1997 Electrophoresis 18:1259-313 (Pub)
Comparison of predicted with
observed protein properties
(localization, postsynthetic modifications)
E.coli
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Computationally checking proteomic data
Property Basis of calculation
Protein charge RKHYCDE (N,C), pKa, pH (Pub)Protein mass Calibrate with knowns (complexes)Peptide mass Isotope sum (incl.modifications)Peptide LC aa composition linear regressionSubcellular Hydrophobicity, motifs (Pub)Expression Codon Adaptation Index (CAI)
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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
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Mr
Cell fraction: Periplasm2D gel:SDS mobility isoelectic pH
Link et al. 1997 Electrophoresis 18:1259-313 (Pub)
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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.
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Isotope calculations
Mass resolution 0.1% vs. 1 ppm
Symbol Mass Abund. Symbol Mass Abund. ------ ---------- ------ ------ ----------- -------H(1) 1.007825 99.99 H(2) 2.014102 0.015 C(12) 12.000000 98.90 C(13) 13.003355 1.10N(14) 14.003074 99.63 N(15) 15.000109 0.37O(16) 15.994915 99.76 O(17) 16.999131 0.038S(32) 31.972072 95.02 S(33) 32.971459 0.75
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Computationally checking proteomic data
Property Basis of calculation
Protein charge RKHYCDE (N,C), pKa, pH (Pub)Protein mass Calibrate with knowns (complexes)Peptide mass Isotope sum (incl.modifications)Peptide LC aa composition linear regressionSubcellular Hydrophobicity, motifs (Pub)Expression Codon Adaptation Index (CAI)
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HighPerformanceLiquidChromatography
trypsin
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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.
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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.
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Empirical linear regression varies with type of LC-material
-NH3+? C18 no yes noW 10.1 9.3 9.8F 8.8 5.5 8.8L 7.5 4.6 9.5I 5.8 3.0 8.4M 4.8 3.0 2.6Y 4.5 3.1 6.1V 3.5 1.3 4.9C 3.4 2.9 0.5P 2.7 0.7 2.8E 0.3 0.5 0.8A 0.2 0.1 1.7D 0.0 0.6 1.1G 0.0 0.0 0.4T -0.1 1.0 1.8S -0.8 -0.1 0.3Q -0.9 0.0 -0.7N -3.0 -2.1 0.0R -3.1 -2.1 2.4H -3.3 -1.5 0.6K -3.5 -1.6 0.0
RP-LC
calculated
observed
Sereda, T. et al. “Effect of the α-amino group on peptide retention behaviour in reversed-phase chromatography.
Wilce, et al. “High-performance liquid chromatography of amino acids, peptides and proteins.” Journal of Chromatography, 632 (1993) 11-18.
(The calculated curve is displaced upward for clarity)
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RT
m/z
min
PSW
CMV
AR
CC
TKDQ
G
AG
L FEK
First Dimension: Reverse Phase Chromatography Separation By Hydrophobicity
Second Dimension: Mass Spectrometry Separation by Mass
A Map is Like a 2D Peptide Gel
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What Information Can Be Extracted From A Single Peptide Peak
m/z
abu
nd
ance
Isotopic Variants of DAFLGSFLYEYSR
0 X 13C
1 X 13C
2 X 13C
3 X 13C
m/z
rt
abu
nd
ance
@ 36.418 min
K.Leptos 2001
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Link, et al. 1999, Nature Biotech. 17:676-82. (Pub)
Directed Analysis ofLarge Protein
Complexesby 2D separation:
strong cation exchangeand reversed-phased
liquid chromatography.
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>95-92-41#peptides
#uniquely identified / #genes
1/1 2/2 1/2 0/2
A new 40S
subunitprotein
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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
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The Finnigan LCQ: An ESI-QIT Mass Spectrometer
Electro-Spray Ionization chamber
Mass Analyzer/Detector
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Tandem Mass Spectrometry
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.
Quadrople Q1 scans or selects m/z. Q2 transmits those ions through collision gas (Ar).Q3 Analyzes the resulting fragment ions.
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Ions
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Peptide Fragmentation and Ionization
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26Gygi et al. Mol. Cell Bio. (1999)
Tandem Mass Spectra Analysis
y
b
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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.
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A dynamic programming approach to de novo peptide sequencing via tandem mass spectrometry
Chen et al 2000. 11th Annual ACM-SIAM Symp. of DiscreteAlgorithms pp. 389-398.
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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)
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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
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Expression quantitation methods
RNA Protein
Genes immobilized labeled RNA Antibody arraysRNAs immobilized labeled genes- Northern gel blot WesternsQRT-PCR -none-Reporter constructs sameFluorescent In Situ (Hybridization) same (Antibodies)Tag counting (SAGE) -none-Differential display mass spec
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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
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Yeast Protein ESI-MS Quantitation
y = 0.8754x + 0.1573
R2 = 0.8381
0.1
1
10
100
1000
10000
0.1 1 10 100 1000
Day 1 measure
Da
y 2
Me
as
ure
Link, et al
MS Protein quantitation R=.84
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Coefficients of Variance
0
1
2
3
4
5
CV
Fre
qu
ency
Sample: Angiotensin, Neurotensin, Bradykinin
Map: 600 – 700 m/z
CV =
MS quantitation reproducibility
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Correlation between protein and mRNA abundance in yeast
Gygi et al. 1999, Mol. Cell Biol. 19:1720-30 (Pub)
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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)
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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 2n=4 6 4 3 3
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Correlation of (phosphorimager 35S 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
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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
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40Gygi et al. Nature Biotechnology (1999)
ICAT Strategy for
Quantifying Differential
Protein Expression.
X= H or D
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41Gygi et al. Nature Biotechnology (1999)
Mass Spectrum and
Reconstructed Ion
Chromatograms.
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Protein & mRNA Ratios +/- Galactose
Ideker et al 2001
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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
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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
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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)
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Natural crosslinks
Disulfides Cys-Cys Collagen Lys-Lys
Ubiquitin C-term-Lys Fibrin Gln-Lys
Glycation Glucose-LysAdeno primer proteins dCMP-Ser
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Crosslinked peptide Matrix-assisted laser desorption ionization Post-Source Decay (MALDI-PSD-MS)
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 E45-R60 (M + H+ = m/z 2059.08).
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Constraintsfor homology modeling based on MS
crosslinking distancesThe 15 nonlocal throughspace distance constraints generated by the chemical cross-links (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)
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Homology modeling accuracy
20
30
40
50
60
70
80
90
100
1 1.5 2 2.5 3 3.5 4
Series1% sequenceidentity
Swiss-model RMSD of the test set in Angstroms
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Top 20 threading models for FGF ranked by crosslinking constraint error
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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
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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)
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1634 Metabolite Masses256 amino acids
0
200
400
600
80 320 560 800 1040 1280 1520
Frequency
Karp et al. (1998) NAR 26:50. EcoCyc; Selkov, et al. (1997) NAR 25:37. WITOgata et al. (1998) Biosystems 47:119-128 KEGG
Databases
598 have identical masse.g. Ile & Leu = 131.17
160 240
Y=
X = Mass
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Y= RPLCretentiontimein min.(higherhydro-phocity)
X = Mass
IL
W
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Metabolite fragmentation &
stable isotope labeling
Wunschel J Chromatogr A 1997, 776:205-19 Quantitative analysis of neutral & acidic sugars in whole bacterial cell hydrolysates using high-performance anion-exchange LC-ESI-MS2.(Pub)
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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 cycles... 13C (or 14C) labeled substrates."
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MetaFoR: Metabolic Flux Ratios
Fractional 13C labeling > Quantitative 2D NMRWhy 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
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A functional genomics strategy that uses metabolome data to reveal the phenotype of
silent mutations
Raamsdonk et al. 2001 Nature Biotech 19:45.
-40C MeOH> 80C EtOH > Cobas Enzymatic BioAutoanalyser & Quantitative 1H NMR 0 to 4.4 ppm (1300 measures)
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Types of interaction modelsQuantum Electrodynamics subatomicQuantum mechanics electron cloudsMolecular mechanics spherical atoms (101Pro1)Master equations stochastic single molecules (Net1)
Phenomenological rates ODE Concentration & time (C,t)Flux Balance dCik/dt optima steady state (Net1)Thermodynamic models dCik/dt = 0 k reversible reactions
Steady State dCik/dt = 0 (sum k reactions) Metabolic Control Analysis d(dCik/dt)/dCj (i = chem.species) Spatially inhomogenous models dCi/dx
Increasing scope, decreasing resolution
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How do enzymes & substrates formally differ?
ATP E2+P ADP E EATP EP
E
A EA EB B
Catalysts increase the rate (&specificity) without being consumed.
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Enzyme rate equations with one Substrate & one Product
dP/dt = V (S/Ks - P/Kp)
1 + S/Ks + P/Kp
ES P
As P approaches 0:
dP/dt = V
1+ Ks/S
S
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Enzyme Kinetic Expressions
Phosphofructokinase
4
6
4
44
0
6
6
611
11
1
161
6
PFKPF
PFKAMP
PFKMg
PFKATP
free
PFKPFK
PFKATPMg
PFKATPMg
PFKPF
PFKPF
PFK
PFKmx
PFK
KPF
KAMP
KMg
KATP
LN
KATPMg
KATPMg
KPFK
PF
N
vv
Allosteric kineticparameters for AMP, etc.
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Human Red Blood CellODE model
GLCe GLCi
G6P
F6P
FDP
GA3P
DHAP
1,3 DPG
2,3 DPG
3PG
2PG
PEP
PYR
LACi LACe
GL6P GO6P RU5PR5P
X5P
GA3P
S7P
F6P
E4P
GA3P F6P
NADPNADPH
NADPNADPH
ADPATP
ADPATP
ADP ATPNADHNAD
ADPATP
NADHNAD
K+
Na+
ADP
ATPADP
ATP
2 GSH GSSGNADPH NADP
ADO
INO
AMP
IMPADOe
INOe
ADE
ADEeHYPX
PRPP
PRPP
R1P R5PATP
AMPATP
ADP
Cl-
pH
HCO3-
ODE model
Jamshidi et al.
2000 (Pub)
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Red Blood Cell in Mathematica
ODE model
Jamshidi et al.
2000 (Pub)
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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