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General FAQ about Mass Spectrometry
GENERAL FAQ ABOUT Mass Spectrometry
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Contributed by Walt
McMurray, Ph.D., Co-Director, YCC/Keck MS Resource
What is an "exact mass" and why
is it useful?
What is an "ion source"?
What is the minimum mass difference needed to distinguish two approximately
100 KD proteins?
What is neutral loss?
What is a quadrupole mass spectrometer?
What does a resolving power of 2,000 mean?
What is a reflectron and what does it do?
What is an "exact mass" and why
is it useful?
The exact mass can be used to confirm an elemental composition (i.e., the
number of carbons, hydrogens, nitrogens, etc) in a reasonably small molecule
(i.e., less than about 1,000 Da on the Micromass Q-Tof API in the Keck
Laboratory) produced perhaps from the chemical synthesis of a potential
chemotherapy drug. In this instance, one calculates the theoretical "exact mass"
by summing up the masses of all the elements expected to be in the compound.
This calculated mass is then compared to the experimentally measured mass to
determine if they agree within the expected 5 parts per million (ppm)
specification of the Q-Tof API - which generally would be acceptable for
publication. Measuring the exact mass in these cases is relatively easy because
one knows what the answer should be before beginning the mass
spectrometry. The reverse process is more difficult. In this case the elemental
composition of the sample is not known so one begins by measuring the mass of
the sample. Now the working assumption must be made that the actual mass is
within the average error (5 ppm) - which it may not be - remember there will be
some mass measurements whose errors are going to be larger than the average
mass error. There are computer programs which will calculate all
elemental compositions which fit the measured mass within the expected error or
one can use a wider error window. The problem is that when the molecule contains
additional elements (e.g., sulfur, phosphorus, silicon or if a large number of
nitrogens and oxygens are allowed in the computation of possible formulas) there
will be several elemental formulae that will fit within the +/-5 ppm
average error
range. This is why Smith (Conrads, T. P., Anderson, G. A., Veenstr, T. D.,
Pasa-Tolic, L., and Smith, R. D., "Utility of Accurate Mass Tags for
Proteome-Wide Protein Identification,", Anal. Chem., 2000, 72, 3349-3354) cites
1 ppm error as being needed for "accurate mass tags" for peptide identification.
What is an "ion source"?
To be analyzed by a mass spectrometer a molecule has to be converted to an
ion. This is done in the ion source. There are many types of ion sources but for
biological (i.e., polar) samples like peptides, proteins, and oligonucleotides
the most useful sources are matrix assisted laser desorption ionization (MALDI)
and electrospray ionization (ESI). In the case of MALDI, the biomolecule is
dissolved in a solution containing a matrix and then deposited onto a target
plate and dried. The crystallized matrix containing the analyte is irradiated
with a laser, typically a nitrogen laser operated at a wavelength of 337 nm. It
is generally thought that the chromophoric matrix absorbs the incident light
energy which expands into the gas phase carrying the analyte molecule with it. A
proton is transferred from the matrix molecule to the analyte producing the
singly charged ion. The selection of the matrix is very important to the success
of the analysis. Electrospray ionization is the method used with the Q-Tof API
in the Keck Laboratory and this is the technique used for liquid
chromatography/mass spectrometry (LC/MS). It is a solution based method of
forming ions, with a typical solution being 50% acetonitrile/ 0.1% formic acid
but chloroform/methanol or other volatile solvents can be used. It is easiest to
think of electrospray as an aerosol (i.e. a stream of tiny droplets) where there
is an applied voltage (1- 3kV) between the source of the spray and the target of
the spray (the sampling cone), The polarity of the applied voltage determines
whether positive or negative ions are produced. The spray droplets contract in
volume as the solvent volatilizes. As the droplet contracts depending on its
size it will either split up into smaller droplets due to electrostatic
repulsion or, if the droplet is very small the electric potential on the surface
of the drop becomes so large that the positively charged molecules are ejected
from the surface of the droplet. These ejected, positively charged molecules are
attracted to the sampling cone by the potential difference and the vacuum which
is at the entrance to the mass spectrometer. The ions travel through one or two
stages of vacuum reduction prior to entrance into the first mass spectrometer,
the quadrupole. On the Q-Tof the quadrupole is followed by a hexapole which is
the collision cell and then by a time-of-flight (TOF) analyzer.
What is the minimum mass difference needed to distinguish two approximately
100 KD proteins?
The biggest limitation in resolving two 100 kD proteins is the width of the
isotope profile which at 100 kD is approximately 50 mass units wide (Yergey, J.,
Heller, D., Hansen, G., Cotter, R. J., and Fenselau, C., Anal. Chem.1983, 55,
353-356). Therefore to detect two approximately 100 kD proteins the mass
difference between the two proteins should be at least 50 Da. This mass
difference can be detected with an instrument resolving power of ~ 2,000 which,
for instance, is within the instrument resolving power of about 10,000 on the
Micromass Q-Tof API in the Keck Laboratory. Note also the subtle difference
between the term resolution; which is the smallest separation, in m/z or dalton,
between two peaks such that they can be distinguished; versus resolving power,
which is m/Dm, in which Dm is the line width at 50% maximum peak height. Another
factor that needs to be considered is that to detect the smallest difference in
mass, the concentrations of the two proteins should be similar. Another
challenge that commonly occurs is that proteins of this size may not be
homogeneous (e.g., they may have micro-heterogeneity due to differential levels
and types of posttranslational modifications or they may contain sodium or other
cationic adducts). This results in broader peaks and peaks with "tails" on them
which means a larger mass difference is required to detect the two components.
The mass accuracy on the Q-Tof API is about +0.02% or less (or about +20 Da) for
a homogeneous 100,000 Da protein. Hence, if one had a solution of a 100,000 Da
protein where half of the protein molecules had been modified with a
posttranslational modification, the latter could be resolved and mass measured
if it introduced a mass difference greater than about 50 Da . However, the
uncertainty in the mass measurements (+/-20 Da in the measurement of each
component) would prevent the identification of the modification.
What is neutral loss?
This expression refers to loss of an uncharged fragment from a
molecule. Generally; the neutral molecule is acetic acid, phosphoric acid or
some other low molecular weight molecule. The neutral molecule is formed when an
acetyl or phosphate group extracts a hydrogen from some site on the intact
molecule. This process takes place in the collision cell (it can also occur in
the nozzle-skimmer region (where the spray enters the ion source)). The neutral
loss of 98 (H3PO4) can be used to detect phosphopeptides because the neutral
loss fragmentation takes place at lower energy than that required to fragment
the peptide backbone.
What is a quadrupole mass spectrometer?
A quadrupole mass spectrometer consists of four parallel rods oriented like
the four poles on a compass. The North-South pair have a radio frequency (rf)
and direct current (dc) voltage of one polarity and the East -West pair have rf
and dc voltages of opposite polarity. Hexapoles and octapoles are similar but
have six and eight rods respectively. They are used as collision and/or storage
cells. In tandem mass spectrometers such as triple quadrupoles or Q-Tofs the
quadrupole functions as either a mass spectrometer or in an ion transmission
mode. To acquire MS spectra the quadrupole is operated in the so-called rf mode
in which it acts to transmit the ions from the ion source to the third
quadrupole or the TOF where the spectrum is recorded. In daughter ion/product
ion MS/MS mode the quadrupole is used in a static mode to select a particular
ion for CAD (collision activated dissociation). The resulting MS spectrum can be
used to determine a partial amino acid sequence of a peptide. In parent
ion/precursor ion MS/MS and neutral loss MS/MS the quadrupole is scanned in a
particular relationship to the third quadrupole or the TOF. Both these types of
scans may be used to detect phosphopeptides
What does a resolving power of 2,000 mean?
In its simplest form it means one can distinguish the peak at mass 2,000
from a peak at mass 2,001. There are more formal definitions of resolution which
is M/Dm where M is the mass where the resolution is measured and delta m is the
mass difference between two peaks of equal magnitude in which the valley between
the peaks is 10%. The mass resolving power is defined for a single peak in which
Dm is the full width of the peak at 50% of the height of the peak (i.e., the
full width at half maximum (FWHM)). One advantage of the 50% definition is that
then the definition of resolving power is the same as resolution (i.e., two
peaks separated by one linewidth are barely resolved). For a discussion of
resolution, resolving power, mass accuracy and mass precision see Marshall,
A.G., Hendrickson, C. L., and Shi, S., D.-H., Anal. Chem., 2002, 74, 252A-259A.
What is a reflectron and what does it do?
A reflectron is a series of electrical fields which act to retard the
approaching ion, then turn it around and accelerate it in the general direction
from which it arrived. Because ions formed in the ion source have a distribution
of kinetic energies when they leave the ion source, ions of the same mass will
not arrive at the detector at the same time. The reflectron compensates for this
distribution of energy by allowing the faster ions to penetrate farther into the
reflectron than the slower ions. With the properly set electric fields on the
return trip the faster ions will catch up to the slower ions just as they
reached the detector. The reflectron reduces the kinetic energy spread of the
ions which results in improved resolution and mass accuracy. |
