Gráficos y mapas de influencia-dependencia de variables en la carrera de Ingeniería

Gel electrophoresis is the most convenient method to visualise quickly the macromolecular content of a sample. Proteins are separated by virtue of their size using polyacrylamide gel electrophoresis (PAGE) with sodium dodecyl sulphate (SDS). The proteins migrate at a rate inversley proportional to their molecular masses under the influence of an electric current and are visualised by a number of methods. Isoelectric focussing is a technique whereby the isoelectric point of the proteins in a sample may be visualised. In Western blotting the proteins from the gel can be electrophoretically transferred to a membrane and probed with specific antibodies for identification. Visualisation is achieved by the use of a second antibody enzyme conjugate that catalyses a colour change in a substrate. Blotting can be employed to identify specific properties of the proteins such as glycosylation. Further characterisation may require amino acid sequence determination and disulphide mapping. An important aspect of the characterisation is the functionality of the protein preparation. This would ideally be demonstrated in vitro and in vivo. Spectrophotometric analyses of the protein sample may be useful to determine the purity and concentration.

2.3.1 Mass spectrometry

Mass spectrometers use the difference in mass-to-charge ratio (m/z) of ionized atoms or molecules to separate them from each other. Mass spectrometry is therefore useful for identification of atoms or molecules and also for determining chemical and structural information about molecules. Molecules have distinctive fingmentation patterns that provide structural information which can be used to identify structural components. The general operation of a mass spectrometer is to create gas-phase ions, to separate the ions in space or time based on their mass-to-charge ratio, and to measure the quantity of ions of each mass-to-charge ratio. The ion separation power of a mass spectrometer is described by the resolution, which is defined as:

m

R = Equation 2.1

om

a mass spectrum (e.g. a mass spectrometer with a resolution of 1000 can just resolve an ion with a m/z of 100.0 from an ion with a m/z of 100.1).

2.3.2 Instrumentation

In general a mass spectrometer consists of an ion source, a mass-selective analyser, and an ion detector. Since mass spectrometers create and manipulate gas-phase ions, they operate in ahigh-vacuum system. The magnetic-sector, quadrupole, and time-of-flight designs also require extraction and acceleration ion optics to transfer ions from the source region into the mass analyser. The details of mass analyser designs and basic descriptions of sample introduction and ionization and ion detection are discussed below.

2.3.3 Electron ionization (El) and chemical ionization (Cl)

Electron ionization (El) is widely used in mass spectrometry for relatively volatile samples that are insensitive to heat and have relatively low molecular mass. An El source uses an electron beam, usually generated from a tungsten filament, to ionize gas-phase atoms or molecules. The spectra, usually containing many fragment-ion peaks, are useful for structural characterisation and identification, and small impurities in the sample are easy to detect. Chemical ionization (Cl) may be used to enhance the abundance of a chosen molecular ion and can be applied to similar samples as EL For both ionization methods, the molecular mass range is 50 to 800 Da. In rare cases it is possible to analyse samples of higher molecular mass. Chemical ionization (Cl) uses a reagent ion to react with the analyte molecules to form ions by either a proton or hydride transfer. The reagent ions are produced by introducing a large excess of methane (relative to the analyte) into an electron impact (El) ion source.

2.3.4 Electrospray ionization (ESI)

Electrospray ionization (ESI) allows production of molecular ions directly from samples in solution. The ESI source consists of a very fine needle and a series of skimmers. A sample solution is sprayed into the source chamber to form droplets. The droplets carry charge when they exit the capillary. As the solvent evaporates, the droplets disappear leaving highly charged analyte molecules. ESI is particularly useful for large biological molecules that

are difficult to vaporize or ionize. It can be used for small and large molecular-mass biopolymers (peptides, proteins, carbohydrates, and DNA fragments), and lipids. The sample must be soluble, stable in solution, polar, and relatively clean (free from nonvolatile buffers, detergents, salts). Unlike MALDI, which is pulsed, it is a continuous ionization method that is suitable for using as an interface with HPLC or capillary electrophoresis where multiply charged ions are usually produced. ESI should be considered a complement to MALDI.

2.3.5 Laser ionization (LIMS)

A laser pulse can ablate material from a surface and create a microplasma that ionises some of the sample constituents. The laser pulse accomplishes both vaporization and ionization of the sample.

2.3.6 Matrix-assisted laser desorption ionization (MALDI)

Matrix-assisted laser desorption (MALDI) is a LIMS method of vaporizing and ionising large biological molecules such as peptides, proteins, oligonucleotides, and other compounds of biological origin as well as of small synthetic polymers. The macromolecule is typically dispersed in a crystal of a matrix material (Table 2.3). MALDI ion sources all consist of a sample stage (or probe) that is used to carry the analyte into the vacuum system of the spectrometer. A UV laser pulse ablates the matrix which carries some of the large molecules into the gas phase in an ionized form so that they can be extracted into the mass spectrometer. Commercial instruments use laser light in pulses of less than 10ns from either a nitrogen laser (337 nm) or a g-switched neodymium: yttrium aluminium garnet (Nd: YAG) laser (226 or 354 nm). Empirical evidence has suggested that the mass analyser and detectors work best when the laser energy is near the threshold level required to produce desorption of the macromolecule (Beavis and Chait, 1996). The matrix material is selected for the type of macromolecule under investigation. Most matrix compounds produce satellite signals called adduct peaks at slightly higher mass than the analyte molecule peaks. These result from the photochemical breakdown of the matrix into a more reactive species which can add to the analyte. The best matrices have low intensity photochemical adduct peaks.

MALDI has enabled the analysis of large biomolecules that were previously only amenable to study using conventional techniques (SDS-PAGE, etc.). The amount of sample needed is very low (pmoles or less). The analysis can be performed in the linear mode (high mass, low resolution) up to a molecular mass o f300,000 (in rare cases) or reflectron mode (lower mass, higher resolution) up to a molecular mass o f10,000 and the analysis is relatively insensitive to contaminants. The mass accuracy (0.1 to 0.01%) is not as high as for other mass spectrometry methods.

2.3.7 Time-of-flight mass spectrometry (TOF-MS)

A time-of-flight mass spectrometer (TOF-MS) uses the differences in transit time through a drift region to separate ions of different masses (Figure 2.2). It operates in a pulsed mode so ions must be produced or extracted in pulses. An electric field accelerates all ions into a field-free drift region with a kinetic energy of q V, where q is the ion charge and Fis the applied voltage. Since the ion kinetic energy KE is O.Smv^ (mass m and velocity v), lighter ions have a higher velocity than heavier ions and reach the detector at the end of the drift region sooner:

K E = q V Equation 2.2

— mv^ = q V Equation 2.3

V = — . Equation 2.4

m

The transit time {t) through the drift tube is L/V where L is the length of the drift tube. / I—

2qV, Equation 2.5

Table 2.3 Properties of MALDI matrices (from Beavis and Chait, 1996). Matrix Analytes* Peptides Proteins Suggested Solvent (water; organic) Ionization ^ Adduct ^ gentisic acid, 2,5- dihydroxybenzoic acid + +/- 9:1 + M + 136 sinapic acid, sinapinic acid, trans-3,5- dimethoxy-4- hydroxycinnamic acid +/- 0 2:1 + M + 206 3-indoleacrylic acid + + 2:1 ++ M + 185 4-HCCA, a-cyano- 4-hydroxycinnamic acid + + 2:1 +++ Footnotes:

^ +, Matrix may be used for most peptides and proteins; +/-, matrix may (or may not) work. ^ The more + signs, the more intense the signal and the higher charge state of the most intense peak.

laser beam