X-Ray Crystallography Laboratory at UTMB
X-Ray Crystallography
at the Sealy Center For Structural Biology
Mass Spectroscopy
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Instrumentation:
MicroMass ElectroSpray
Electrospray
Overview
Electrospray (ES) mass spectrometry (MS) provides a rapid and accurate means of analyzing a wide range of polar molecules. The origin of electrospray can be traced back to 1917 (J. Zeleny, Phys. Rev. 10, 1, 1917) although the first combined ES-MS results were announced in 1984 (J. Fenn, J. Phys. Chem., 88, 4451, 1984), and its first application to protein analysis came four years later from the same group (C.K Meng, M. Mann, J.B. Fenn, Proc. 36th. ASMS, San Francisco, 1988).
For smaller molecules up to 1000 Daltons in molecular mass, either an [M+H]+ or an [M-H]- ion is detected generally depending on whether positive or negative ion detection has been selected. Some fragmentation may be apparent, and indeed can often be induced to provide structural information.
Molecules with higher molecular masses up to 200,000 Daltons usually produce a series of multiply charged ions which can be processed by the data system to give a molecular weight profile with a mass accuracy of ± 0.01%.
Low Molecular Weight Analysis
Polar compounds of low molecular weight(<1000amu) will generally form singly charged ions by the loss or gain of a proton. Basic compounds, e.g. amines, can form a protonated molecule ([M+H]+) which can be analyzed in positive ion mode to give a peak at m/z M+1. Acidic compounds, e.g. sulphonic acids, can form a deprotonated molecule ([M-H]-) which can be analysed in negative ion mode to give a peak at m/z M-1. As electrospray is a very 'soft' ionization technique there is usually little or no fragmentation and the spectrum contains only the quasimolecular ion. Care should be taken in the interpretation of electrospray spectra when the analysis has taken place in the presence of additives or contaminants such as ammonium or sodium ions. Some compounds are susceptible to adduct formation with ions present in solution and may give ions other than the protonated molecule. Common adducts are with NH4+(M+18), Na+ (M+23)and K+ (M+39).
Interpretation of Multiply-Charged Electrospray Spectra
The singly-charged [M+H]+ and [M-H]- ions arising from samples of relatively low molecular masses can be interpreted directly as with other soft ionization techniques such as chemical ionization, thermospray and Fast Atom Bombardment. However, unique to electrospray, the high electric field present has the effect of producing spectra which often show a predominance of multiply-charged ions. Since a mass spectrometer analyses for mass to charge ratio (m/z) these ions appear on the mass spectrum at apparent mass values of :
M+nH = m
n z (1)
where M= actual mass
n= number of charges
H=mass of one proton
This feature of electrospray effectively extends the mass range of the mass spectrometer being used. A sample of horse heart myoglobin (molecular mass 16951.48) produces a positive ion ES spectrum. This consists of a series of multiply charged ions over the range m/z 700-1700, and was acquired on mass spectrometer with a mass range of 4000 Daltons. If only a singly-charged ion had been produced this would have been well outside the range of the instrument. The ion at m/z 942.7 corresponds to 18 positive charges. If the charge state was known the molecular mass could be calculated as follows:
M=(18x942.7) - 18=16950.5
In general the charge state on any particular ion is not known, but any two adjacent ions in the series of multiply charged ions differ by one charge. So in the spectrum shown where two masses have been labelled M1 and M2, it can be said that
n1 = n2 + (2)
where n1 is the number of charges on M1 and n2, is the number of charges on M2. From equation (1) it can be said that
M2 = M+n2H (3)
n2
and M1= M+n1H (4)
n1
Substituting for (2) in (4) gives
M1= M+(n2+1)H (5)
n2+1
Rearranging the two simultaneous equations (3)and (5) gives respectively
n2M2=M + n2H (6)
(n2+1) M1=M + (n2+1)H (7)
and substituting for M in (7) gives
(n2+1)M1=n2M2 - n2H + (n2+1)H (8)
Therefore
n2M1 + M1=n2M2 + H (9)
and
n2= M1-H (10)
M2-M1
The value of n2 is rounded to the nearest whole number. Once the value of n is known the original mass can be calculated from (1), i.e.
M =n2 (M2 - H)
Example.
Any adjacent pair of ions can be taken from the spectrum of horse heart myoglobin to calculate its molecular mass. Using (10),
n2= ???998.1-1.0???
1060.5-998.1
=15.98 (i.e.16)
That is, the ion at 1060.5 equates to the molecule with 16 positive charges on it.
From (1), M= 16(1060.5-H)
=16952.0
MassLynx software calculates one value of n and M and then predicts where other members of the same series of multiply charged ions will occur. The value of M quoted as a result is the average value calculated from each pair of peaks in the series, together with their standard deviation. Other processing techniques can also be used: the m/z spectrum can be transformed to produce a molecular weight profile from which accurate (±0.01%) masses can be read directly, and /or processed by Maximum Entropy techniques to achieve optimum resolution.