What is the origin of the conformation dependence of protein CSDs

1
What is the origin of the conformation dependence
of protein CSDs?
Coulomb repulsions (Distances) Fenn et al.
1990 Mass Spectrom. Rev. 9, 37-70
Solvent accessibility (Schielding) Chowdhurry et
al. 1990 J. Am. Chem. Soc. 112, 9012-9013

• Droplet charge
• (Shape, Size)
• de la Mora 2000
• Anal. Chim. Acta. 406, 93-104

• Coulomb attractions
• (Interactions)
• Grandori 2003
• J. Mass Spectrom. 38, 11-15

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2
Unfolded proteins in ESI-MS (POS)
(Correlation with acidic residues in neg ESI)
(Loo et al. 1990, Anal. Chem. 62, 693-698)
3
The Coulomb-repulsion hypothesis
Physiological conditions
ESI-MS
d

N(n)


U

DG


N
U(n)
d

• Prediction of ionization-induced unfolding
• Assumption of only repulsive electrostatic
  interactions
• Calculated gas-phase basicity for folded proteins
  does not fit exper.

4
Proton-transfer reactions in the gas phase
MHnn H MHn1(n1)
Gas-phase basicity (MHnn) GB - DG Proton
affinity (MHnn) PA - DH
5
Apparent gas-phase basicity of folded proteins
higher than predicted based on Coulomb repulsions
calculated (extended)
(Williams 1996, J. Mass Spectrom. 31, 831-842)
measured
calculated (native)
calculated (helix)
ESI-MS under non-denaturing conditions
6
The solvent-accessibility hypothesis
Lys
His
Glu?
Asp
His?
Arg?

• 30 of ionizable side chains buried or
  partially buried
• Influence of conformation on pKas

7
Influence of protein size
(van den Heuvel Heck 2004, Spectroscopy, 16,
6-13)
8
Effect of surface tension
(Iavarone Williams 2003 J. Am. Chem. Soc. 125,
2319-2327)
9
Investigating the role of surface tension
Main
The Rayleigh equation
Max CS between 70 and 120 of ZR (water)
qR zRe 8p(eogR3)1/2
Max
e 1.6x10-19 Coulomb eo 8.85x10-12
Coulomb2/Nm2
Relevant physico-chemical features of the
solvents employed in this work
10
pH 2.2 (6 mM HCL)
pH 2.2 (10 1.7 M acetic acid)
Lysozyme (folded)
Cytochrome c (unfolded)
Myoglobin (unfolded)
(amalikova et al. 2004, Anal. Bioanal.
Chem. 378, 1112-1123)
11
Ubiquitin (folded)
Lysozyme (folded)
Cytochrome c (folded)
17
Cytochrome c (unfolded)
Myoglobin (unfolded)
(amalikova Grandori JACS 125, 13352-13353)
- 1-prOH
1-prOH
12
Mb pH 2.2 (HCOOH)
Cyt c pH 2.2 (HCOOH)
No alcohol
50 1-prOH
50 2-prOH
Intensity
13
Lyz
Ubq
(water)
20 1-prOH
20 2-prOH
50 1-prOH
50 2-prOH
14
(No Transcript)
15
Reciprocal charge stabilization in folded proteins
ESI (positive-ion mode)
Solution pH 7
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Amino acids, peptides and unfolded proteins
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Folded proteins


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16
Charge neutralization during ESI
Positive-ion mode
X-COO_ H3O gt X-COOH H2O
(Frequent involvement of ammonium acetate)
Negative-ion mode
X- NH3 OH_ gt X- NH2 H2O
17
100
Folded
Intensity
Unfolded
m/z
0
2000
500
Stability in vacuo
Native-like ions
Neutralization products
NO
Calculated low GBapp
Extensive ionization
Limited ionization
18
Role of negative charges in positive-ion mode
(Ang II in water)
NRVYVHPF DRVYIHPF


3
100
2
Intensity
0
300
600
m/z
(amalikova Grandori 2003, J. Mass Spectrom.
38, 11-15)
19
Detect different charge states of homologous
proteins (nano-ESI-MS in water)
10
12
Horse Mb Sperm-whale Mb
100
Intensity
0
1500
500
m/z
20
RNase mutants in negative-ion mode(nano-ESI-MS
pH 6.5)
6-
6-
WT
D17K
100
100
Intensity
0
0
500
4000
500
4000
5-
6-
D1K D17K E41K
D17K E41K
100
100
Intensity
0
0
500
4000
500
4000
m/z
m/z
21
RNase mutants in negative-ion mode(nano-ESI-MS
pH 6.5)
Intensity (counts per spectrum)
22
RNase mutants in positive-ion mode(nano-ESI-MS
in water)
8
5K (5)
8
4K (3)