1st FECS school on protein chemistry

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1st FECS school on protein chemistry

• Introduction to MS
• MS of proteins
• MS in proteomics

Rita Grandori 13 Jan 2005 Brixen, Italy
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MS applications in Protein Science

• Protein identification
• Protein sequencing
• Posttranslational modifications
• Tridimensional structure
• Protein folding
• Non-covalent complexes
• Imaging

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The main steps of MS measurements

• Generate ions of the analyte in the gas phase
• Sort ions on the basis of their m/z value
• Count ions

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The main elements of a mass spectrometer
SOURCE
ANALYZER
DETECTOR
(gas-phase ions)
(ion sorting)
(ion detection)
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Ionization methods
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MALDI and ESI spectra of RNase
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MALDI-MS RNase A
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Intensity (arbitrary units)
ESI-MS RNase Sa
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Mass deconvolution (positive-ion mode ESI-MS
cytochrome c pH 2.2)
i
j
(m/z)i(mpmazi)/zi (m/z)jmpma(zi-1)/(zi-1
)
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MALDI/ESI comparison
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MALDI
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MALDImatrices
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The ESIprocess
5 kV (1.5 kV for nano-ESI)
Kebarle 2000 J. Mass Spectrom. 35, 804-817
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Charged-droplet fission during ESI
Kebarle Ho 1997, in Electrospray ionization
mass spectrometry (Wiley)
N charges per droplet
R droplet radius (mm)








qR Rayleigh-limit charge e elementary charge
(1.6x10-19 Coulomb) e0 Permittivity of
vacuum (8.85x10-12 Coulomb2/Nm2) g
Surface tension of solvent R Droplet radius
qR ze 8p (e0gR3)1/2
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Two alternative models for the production of
gas-phase ions during ESI
The charged-residue model (Dole et al. 1968, J.
Chem. Phys. 49, 2240-2249) Solvent evaporation
from droplets containing one molecule of the
analyte The ion-evaporation model (Iribarne
Thomson 1976, J. Chem. Phys. 64, 2287-2294) Ion
evaporation from charges droplets before
Rayleigh instability
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The nano-spray source
1mm
Smaller droplets gt milder voltage and
temperature conditions 5 ml of 5 mM protein
solution gt 1h measure (3 s/spectrum)
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Mass analyzers
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Quadrupole
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Ion Trap
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Magnetic sector
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TOF

• Orthogonal acceleration
• Acquired kinetic energy
• depends on z
• Drift in field-free flight tube
• Time of flight depends on m
• (at given kinetic energy)

y
x
KEy zeV KEy ½ mvy2
T L(m/2zeV)1/2
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FT-ICR
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Accuracyandresolution
TOF R 5,000-20,000 ( 200,000
Euros) FT-ICR R 300,000 ( 1,000,000 Euros)
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Isotopic resolution
I
m/z
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Isotopic resolution on protein peaks by FT-ICR
Z(Dm/z)-1
R 300,000
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Cytochrome c (horse)

• 103 amino acids
• Basic
• Mainly helical
• Covalently bound heme
• Acid-induced unfolding
• (pH 3-2)

Early-folding subdomain
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Protein folding studies by ESI-MS(same mass,
different charges)
Conformation-dependence of charge-state
distributions
Preservation of non-covalent interactions
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100
Folded
Intensity
Experimental identification of F and U
Cyt c pH 2.8
Unfolded
18
0
2000
500
m/z
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Electrolytic effects during ESI
2H2O gt 4H 4e- O2 (E 1.23 V)
(Konermann et al. 2001, Anal. Chem. 73, 4836-4844)
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Electrochemically induced pH changes
Cyt c in 40 propanol, 1 mM KNO3, pH 5.6
(Konermann et al. 2001, Anal. Chem. 73, 4836-4844)
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No direct effect of pH on CSD(Ubiquitin)
pH 7
pH 2.2 (HCl)
pH 2.2 (HCOOH)
Effect of hydrophobicity of the cosolvent HCL lt
HCOOC lt CH3COOH
pH 2.2 (CH3COOH)
(amalikova et al. 2004, Anal. Bioanal. Chem.
378, 1112-1123)
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No direct effect of pH on CSD(Lysozyme)
pH 7
pH 2.2 (HCl)
Charge-reduction effect
MHnn CH3COO- MHn-1(n-1) CH3COOH
pH 2.2 (CH3COOH)
(amalikova et al. 2004, Anal. Bioanal. Chem.
378, 1112-1123)