ESI and MALDI Mass Spectrometry
S. Sankararaman
Department of Chemistry
Indian Institute of Technology Madras
Chennai 600036
[email protected]
MODULE 22
ESI-Q MASS SPECTROMETRY - examples
Mol. Wt = 1140
[M+H+] (m/z 1141, z=1)
and [M+2H+] (m/z 571, z=2)
are seen
ESI-MS of antibiotic gramicidine
formyl-L-X-Gly-L-Ala-D-Leu-L-Ala-D-ValL-Val-D-Val-L-Trp-D-Leu-L-Y-D-Leu-L-TrpD-Leu-L-Trp-ethanolamine
X = valine or isoleucine and Y = tryptophan
ESI-MS of polyethylene glycol (PEG)
The nomial molecular weight correspond
to the most abundant oligomer of PEG
I and II correspond to mono and doubly
charged ions
ESI-MS of polyethylene glycol (PEG) of
various molecular weight
I, II, III, IV etc correspond to the charge
on the molecular ion
The nomial molecular weight correspond
to the most abundant oligomer of PEG
ESI- mass spectra of protein samoles
Protein samples in acidified water, ethanol
and isopropanol mixture. The adducts are protons
ESI- mass spectra of protein samoles
Protein samples in acidified water, ethanol
and isopropanol mixture. The adducts are protons
Mol. Wt 4260
ESI-MS of a synthetic oligonucleotide – negative ion mode
[M+100H+]
Z = 100
Observed m/z = 1331
ESI-MS of bovine serum albumin dimer.
MW = 133,000
Positive ESI-MS m/z spectrum of the protein
hen egg white lysozyme.
Next slide for calculating M (mass)
and n (the charge on the ion)
Calculating the molecular weight from ESI-MS
If the ions appearing at m/z 1431.6 in the lysozyme spectrum have "n" charges,
then the ions at m/z 1301.4 will have "n+1" charges, (due to protonation)
1431.6 = (MW + nH+)/n and 1301.4 = [MW + (n+1)H+] /(n+1)
These simultaneous equations can be rearranged to exclude the MW term:
n(1431.6) - nH+ = (n+1)1301.4 - (n+1)H+
n(1431.6) = n(1301.4) +1301.4 - H+
n(1431.6 - 1301.4) = 1301.4 - H+
and so:
n = (1301.4 - H+) / (1431.6 - 1301.4)
hence the number of charges on the ions at m/z 1431.6 = 1300.4/130.2 = 10.
Putting the value of n back into the equation:
1431.6 = (MW + nH+) n
gives 1431.6 x 10 = MW + (10 x 1.008)
and so MW = 14,316 - 10.08
therefore MW = 14,305.9 Da
Molecular mass profile of lysozyme obtained by
maximum entropy processing of the m/z spectrum
Proteins in their native state, or at least containing a significant
amount of folding, tend to produce multiply charged ions covering
a smaller range of charge states (say two or three). These charge
states tend to have fewer charges than an unfolded protein would
have, due to the inaccessibility of many of the protonation sites.
In such cases, increasing the sampling cone voltage may provide
sufficient energy for the protein to begin to unfold and create a wider
charge state distribution centering on more highly charged ions in the
lower m/z region of the spectrum.
ESI-MS of native state protein and unfolded protein are different!!
Positive ESI-MS m/z spectra of the protein apo-pseudoazurin
analysed in water at pH7 (upper trace)
and in 1:1 acetonitrile:0.1% aq. formic acid at pH2 (lower trace).
(refer to spectra in the previous slide)
Analysis of the protein in 1:1 acetonitrile : 0.1% aqueous formic acid at pH2
gave a Gaussian-type distribution with multiply charged states ranging from
n = 9 at m/z 1487.8 to n = 19 at m/z 705.3, centering on n = 15 (lower trace).
The molecular mass for this protein was 13,381 Da. Analysis of the protein in
water gave fewer charge states, from n = 7 at m/z 1921.7 to n = 11 at m/z
1223.7, centering at n = 9 (upper trace). Not only has the charge state
distribution changed, the molecular weight is now 13,444 Da which represents
an increase of 63 Da and indicates that copper is remaining bound to the protein.
Many types of protein complexes can be observed in this way, including
protein-ligand, protein-peptide, protein-metal and protein-RNA
macromolecules.
From our lab: identification of macrocycles as Ag+ complex
MeO
MeO
OMe
OMe
n
S. Sankararaman, A. Bandyopadhyay
J. Org. Chem. 2006, 71, 4544-4548.
n
Macrocycle
Yield (%)
1
2
3
4
5
6
7
Dimer
Trimer
Tetramer
Pentamer
Hexamer
Heptamer
Octamer
16
10
33
8
13
2.5
6
MeO
OMe
MeO
OMe
MeO
OMe
OMe
MeO
MeO
NMR is not useful for characterization
due to similar spectra for all
sizes of the macrocycle
OMe
ESI-MS OF Ag+ COMPLEXES OF HEXAMER
calculated
experimental
The cluster of peaks is due to isotopes of Ag+ in the [M+Ag+] adduct
ESI-MS OF Ag+ COMPLEXES OF OCTAMER
calculated
experimental
n
Macro
cycle
HRMS
calc.for
HRMS
found
2
Trimer
C36H30O6Ag
665.1093
665.1112
3
Tetramer
C48H40O8Ag
851.1774
851.1802
4
Pentamer
C60H50O10Ag
1037.2455
1037.2451
5
Hexamer
C72H60O12Ag
1223.3136
1223.3112
6
Heptamer
C84H70O14Ag
1409.3817
1409.3792
7
Octamer
C96H80O16Ag
1595.4497
1595.4481
THANK YOU