Matrix Assisted Laser Desorption/Ionization is a method of transferring large, labile molecules into the gas
phase as intact ions. The technique involves mixing the analyte of interest with a large molar excess of a matrix compound, usually a
weak organic acid. This mixture is placed on a vacuum probe and inserted into the time-of-flight mass spectrometer for laser desorption
analysis. This technique of sample preparation allows for precise and fast molecular weight determination of peptides, proteins,
carbohydrates, nucleic acids, synthetic polymers or other compounds up to several hundred kDa.
In the electrospray ionization (ESI) process, a flow of sample solution is pumped through a narrow-bore metal
capillary held at an electric potential of a few kilovolts relative to a counter electrode. Drops of the solution, emerging from the
tip of the needle, spray as a mist of very fine, charged droplets. As these charged droplets evaporate in a flow of a warm drying gas,
the increasing coulombic repulsive forces exceed the surface tension causing the droplets to undergo fission. Successive fission
ultimately gives rise to droplets containing a single solute molecule that retains the droplet charge left as the remaining solvent
evaporates. Multiply charged ions are commonly observed. This spraying ionization process takes place at atmospheric pressure
Electrospray ionization is a very mild process with little thermal input. ESI is capable of generating multiply
charged ions with low mass/charge ratio, which can be easily analyzed by quadrupole mass spectrometry. Computer-aided algorithms have
been developed to derive molecular weight information from these multiply charged ion series. Averaging the results of multiply charged
peaks leads to mass measurement with an accuracy of ±0.01%. It is often used to measure the molecular weights of proteins and
polypeptides with molecular weights up to ~ 60 kDa.
Electrospray MS can be coupled to high performance liquid chromatography
(HPLC) using a microbore HPLC column or a packed capillary LC column. This reduces tedious sample preparation and sample loss during
preparation. An enzymatic digest of protein can be analyzed by LC/MS for peptide mapping. LC/MS can be used for the quantitative
analysis of trace samples including metabolites or contaminants. LC/MS/MS is almost the best method available to study
pharmacokinetics, the fate of drugs, pesticides and insecticides. Not only can it quantitate the amount of specimen in a mixture but
also elucidate the structures of the specimen.
Fragmentation is virtually absent in electrospray mass spectrometry, but can
be deliberately induced by MS/MS techniques. Tandem MS/MS employs collision induced dissociation
(CID) to fragment a precursor ion. For a sample mixture, the first quadrupole mass analyzer generates a spectrum of ions. The ion of
interest is then selectively transmitted into a collision cell, containing an inert gas, which permits its collision with the selected
ion. The fragmentation is caused by collision-induced dissociation (CID) and resulting ions are then mass analyzed in the final mass
analyzer as product ions. Structural information can be derived from these fragments. Applications of tandem mass spectrometry are
found in the structure elucidation of small organic molecules, carbohydrates, nucleosides, oligonucleotides, fatty acids, lipids, DNA
and RNA adducts, etc.
In the past a few years, electrospray mass spectrometry has been playing an increasing role in determining
peptide sequence. Mass spectrometry provides an efficient approach to identifying a known protein or finding a specific short sequence
of a peptide. A CID mass spectrum will also reveal the location and amino acid position of the posttranslational modification. The
success of tandem MS in protein sequencing is that the CID technique cleaves the peptide bond and generates a series of ions with
increasing masses. The mass difference between consecutive ions reveals the identities of the consecutive amino acids. In electrospray
mass spectrometry, structural information on selected species is usually obtained through collision-induced dissociation on the doubly
charged ions using low collision energy.
The ESI mass spectrometers are compatible with most biological
materials with their molecular weights up to approximately 60 kDa. The MALDI-TOF instrument is less useful at low molecular weights due
to matrix interference, but has a theoretically unlimited mass range. The samples should be soluble in aqueous solvents (water,
methanol, acetonitrile) and prepared with greater than 5 pmol/µL concentration.
The instrument tolerates most volatile buffers such as ammonium acetate or ammonium bicarbonate. The maximum
volatile buffer concentration is 20 mM. Non-volatile agents like salts (e.g., NaCl, KH2PO4, Tris), detergents
(e.g., Tween, Triton, SDS), chaotropic agents (e.g., Urea, Guanidinium salts) and solvents (e.g., DMSO, Glycerol) should be avoided.
High buffer or detergent concentrations will induce ion suppression, which decreases the sensitivity.