Introduction to MS

1
Introduction to MS

• Introduction to MS methods
• The ESI process
• Conformation dependence of protein ESI-MS spectra

2
Mass spectrometry

• Mass spectrometry is a technique that allows
  high-accuracy (20 ppm) determination of the mass
  of gaseous ions on the basis of their
  mass-to-charge (m/z) ratio.
• It is important to appreciate how this simple
  fact can be exploited to gain information that
  goes far beyond molecular mass determination and
  extends to
• Conformational studies
• Binding studies
• Protein identification
• Protein sequencing
• Analysis of post-translational modifications

3
MS applications in Protein Science

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

4
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

5
The main elements of a mass spectrometer
SOURCE
ANALYZER
DETECTOR
(gas-phase ions)
(ion sorting)
(ion detection)
6
Ionization methods
7
MALDI and ESI spectra of RNase
1
MALDI-MS RNase A
2
Intensity (arbitrary units)
ESI-MS RNase Sa
8
MALDI/ESI comparison
9
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
)
10
The ESI-TOF mass spectrometer

• 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
11
Mass analyzers
12
Accuracyandresolution
TOF R 5,000-20,000 ( 200,000
Euros) FT-ICR R 500,000 ( 1,000,000 Euros)
13
The ESIprocess
5 kV (1.5 kV for nano-ESI)
Kebarle 2000 J. Mass Spectrom. 35, 804-817
14
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
15
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
16
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)
17
Cytochrome c (horse)

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

Early-folding subdomain
18
Protein folding studies by ESI-MS(same mass,
different charges)
Conformation-dependence of charge-state
distributions
Preservation of non-covalent interactions
9
100
Folded
Intensity
Experimental identification of F and U
Cyt c pH 2.8
Unfolded
18
0
2000
500
m/z
19
Electrolytic effects during ESI
2H2O gt 4H 4e- O2 (E 1.23 V)
(Konermann et al. 2001, Anal. Chem. 73, 4836-4844)
20
Electrochemically induced pH changes
Cyt c in 40 propanol, 1 mM KNO3, pH 5.6
(Konermann et al. 2001, Anal. Chem. 73, 4836-4844)
21
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)
22
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)
23
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)
• de la Mora 2000
• Anal. Chim. Acta. 406, 93-104

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

_








24
Unfolded proteins in ESI-MS (POS)
(Correlation with acidic residues in neg ESI)
(Loo et al. 1990, Anal. Chem. 62, 693-698)
25
The Coulomb-repulsion hypothesis
Physiological conditions
ESI-MS
d

N(n)


U

DG


N
U(n)
d

• Prediction of ionization-induced unfolding
• (in other words, distances in proteins are free
  to change...)
• Assumption of repulsive electrostatic
  interactions
• Wrong calculated gas-phase basicity for folded
  proteins

26
Proton-transfer reactions in the gas phase
MHnn H MHn1(n1)
Gas-phase basicity (MHnn) GB - DG Proton
affinity (MHnn) PA - DH
27
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
28
The solvent-accessibility hypothesis
Lys
His
Glu?
Asp
His?
Arg?

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

29
Influence of protein size
(van den Heuvel Heck 2004, Spectroscopy, 16,
6-13) (Hautreux et al. 2004, Int. J. Mass
Spectrom. 231, 131-137)
30
Effect of surface tension
(Iavarone Williams 2003 J. Am. Chem. Soc. 125,
2319-2327)
31
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
32
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)
33
Ubiquitin (folded)
Lysozyme (folded)
Cytochrome c (folded)
17
Cytochrome c (unfolded)
Myoglobin (unfolded)
(amalikova Grandori JACS 125, 13352-13353)
- 1-prOH
1-prOH
34
Mb pH 2.2 (HCOOH)
Cyt c pH 2.2 (HCOOH)
No alcohol
50 1-prOH
50 2-prOH
Intensity
35
Lyz
Ubq
(water)
20 1-prOH
20 2-prOH
50 1-prOH
50 2-prOH
36
Reciprocal charge stabilization in folded proteins
ESI (positive-ion mode)
Solution pH 7
_


Amino acids, peptides and unfolded proteins
_




_

_

_
_




Folded proteins


_
_


_
_




37
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
38
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
39
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)
40
Detect different charge states of homologous
proteins (nano-ESI-MS in water)
10
12
Horse Mb expected 9 Sperm-whale Mb expected 12
100
Intensity
0
1500
500
m/z
41
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
42
RNase mutants in negative-ion mode(nano-ESI-MS
pH 6.5)
Intensity (counts per spectrum)
43
RNase mutants in positive-ion mode(nano-ESI-MS
in water)
8
5K (5)
8
4K (3)