General FAQ about Mass Spectrometry

The exact mass can be used to confirm an elemental composition (i.e., the number of carbons, hydrogens, nitrogens, etc) from a reasonably small molecule (i.e., less than <1000 Da) 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 Micro 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.

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 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.

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.

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.

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.

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.

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.