18.3 Protein Identification

281

higher throughput, and post-translational modifications can be readily detected. Mass

spectrometers consist of an ion source, a mass analyser (ion trap, quadrupole, time

of flight (ToF), or ion cyclotron), and a detector.

The objects to be analysed have to be introduced into the MS in the gas phase. This

can be achieved by electrospraying or laser desorption ionization. In electrospray-

ing, the proteins are dissolved in salt-free water, typically containing some organic

solvent, and forced to emerge as droplets from the end of an electrostatically charged

silica capillary. As the solvent evaporates, the electrostatic charge density increases

until the droplets explode. The solution dilution should be such that each protein is

then isolated from its congeners. The remaining solvent evaporates and the protein

molecules pass into the MS. At this stage, each protein molecule is typically multiply

charged. Sequential quadrupole filters, typically three, are used to achieve adequate

discrimination. The mass spectrum for an individual protein consists of a series of

peaks corresponding tom divided by zm/z ratios whose chargezz differs by one electron. The mid-

dle quadrupole may contain a collision gas (e.g., argon) to fragment the protein into

smaller peptides.

In laser desorption ionization, usually called matrix-assisted laser desorption ion-

ization (MALDI) or surface-enhanced laser desorption/ionization (SELDI), the pro-

tein

is

mixed

with

an

aromatic

organic

molecule

[e.g.,

sinapinic

acid,

(CHSubscript 33O)Subscript 22OHCSubscript 66HSubscript 22(CHSubscript 22)Subscript 22COOH] spread out as a thin film, and irradiated by a pulsed

ultraviolet laser. The sinapinic acid absorbs the light and evaporates, taking the pro-

teins with it. Other matrices can be used with infrared lasers. ¹⁴ The proteins are

typically singly charged, and a ToF MS detects all the ions according to their mass.

MALDI-ToF MS cannot detect as wide a range of proteins as quadrupole MS, and

the matrix can exert unpredictable effects on the results. Nevertheless, the vision

of spots on a two-dimensional gel being rapidly and sequentially vaporized by a

scanning laser and immediately analysed in the MS offers hope for the development

of high-throughput proteomics analysis tools.

Newer developments in the field include the application of sophisticated ion

cyclotron resonance MS, the use of Fourier transform techniques, and miniature

instrumentation according to the lab-on-a-chip concept.

Mass spectrometry is also used to characterize the peptide fragments resulting

from proteolysis followed by chromatography. Proteins separated by 2DGE can also

be cleaved using trypsin or another protease to yield fragments, which are then mass-

fingerprinted using MS. The proteolytic peptide fragments are encoded as a set of

numbers corresponding to their masses, and these numbers are compared with a

database assembled from the mass-fingerprints of known peptides.

14 See Chem. Rev. 103 (2003), issue no 2.