Frequently Asked Questions
What is ESI? Electrospray ionization (ESI) is a method for generating molecular ions from samples in solution. ESI is a gentle ionization technique that has been applied to small molecules, like the phospholipids, and large biopolymers (proteins, peptides, oligosaccharides, and oligonucleotides). HPLC, direct infusion, or capillary electrophoresis is used to spray liquid samples directly into the source. The spray tip is maintained at a high potential with respect to ground to ionize droplets in the solvent plume (solvent plus molecules of interest). Molecules are ionized during the desolvation process and are then directed into the mass-filtering region of the mass spectrometer. ESI is not compatible with high salt concentrations, but there are methods and techniques available to minimize challenges posed by salts.
What is Tandem MS? Tandem MS (MS/MS) simply means that two mass analyzers have been coupled. In most instruments another ion containment device is inserted between the two mass analyzers. Ions exiting the first MS collide with heavy, neutral atoms in a containment field and are broken down into smaller fragments. Ion fragments, called product ions, exit into the second MS that separates them based on m/z ratio. This process generates a product ion spectrum (previously called a daughter ion spectrum) that can be used to help identify molecules and sequence peptides.
What is a molecular ion? Adding or subtracting an electron from a neutral molecule (M.- or M+) creates a molecular ion. The mass of the molecular ion is essentially the same as that of the neutral molecule. Gentle ionization methods can protonate a neutral molecule to create a protonated molecular ion (MH+). The molecular weight of the protonated molecule is increased by the weight of one hydrogen atom. Large polymers like peptides and proteins may undergo multiple protonation.
What is the monoisotopic mass? The monoisotopic mass is obtained by summing the isotopic masses of the most abundant naturally occurring stable isotopes of the elements contributing to a molecule. For example, the monoisotopic mass of 1,2-O-octadecanoyl-sn-glycerol-3-phosphocholine (DOPC) is 789.6248 g/mole (for C44H88N1O8P1). Contrast this to the average molecular mass, 790.1584 g/mole, which includes the contributions of all stable isotopes. The nominal mass of a molecule is the mass derived from the sum of the integer masses of the most abundant naturally occurring stable isotopes. For example the nominal mass of DOPC is 789 g/mole.
What does m/z mean?m/z is the mass-to-charge ratio and unitless. Both mass and charge influence the movement of ions through mass analyzers and raw data are m/z. For example the m/z for mono-protonated DOPC is 798.6326. If DOPC could be di-protonated the m/z of the resulting ion would be 395.8202.
What is product ion analysis? To perform product ion analysis a single m/z ratio exiting from the first stage of a tandem mass spectrometer is directed into a chamber containing neutral molecules. The resulting collisions between the ions and the neutral molecules cause the ions to fragment by a process called collision induced dissociation (CID). The product ions are then separated by the second stage of the mass spectrometer to yield a product ion spectrum.
What is precursor ion analysis? Precursor ion analysis, formerly called parent ion analysis, uses the MS/MS capability of tandem mass spectrometers to measure a specific product ion that was formed by CID. Precursor ion analysis is routinely used to quantify classes of molecules that generate unique fragments ion on CID. For example, glycerophosphatidylcholines can be identified in a mixture by the product ion m/z +184.1.
What is common neutral loss analysis?Common neutral loss analysis uses the MS/MS capability of tandem mass spectrometers to identify product ions that were formed by the loss of a neutral fragment on CID. Common neutral loss analysis is used to identify certain classes of molecules that break down with the loss of a specific neutral mass. For example, positively charged glycerophosphatidylethanolamines are identified by the unique loss of 141.1 Da from the precursor ion.
For additional definitions see O. David Sparkman, Mass Spec Desk Reference, Global View Publishing, Pittsburg, PA, 2000.