Capillary Separations Coupled To Ms

While ESI and MALDI have enabled proteins and peptides to be analyzed by MS, the complex mixtures represented by clinical and other biological samples require fractionation prior to analysis. Without fraction-ation, competitive ionization induces only the species that most avidly take up charge to be detected in a sample, resulting in poor sensitivity for many ions; this effect is known as ion suppression. In addition, the mass spectra of unfractionated mixtures can be so complex as to defy interpretation even if extremely high resolution mass spectrometers are used. Resolution of mixtures by a separation technique prior to MS ameliorates these problems. Of the separation techniques available, cLC and CE are two that have received considerable attention for coupling to MALDI-MS.

cLC is comparable to high-performance liquid chro-matography (HPLC), with the main difference being a much reduced internal bore of the column for cLC.

Whereas an HPLC column may have an inner bore of 2-5 mm, a cLC column may have an inner bore of 25300 mm. As mobile phase flow and sample volume requirements scale with the square of inner diameter (i.d.), this miniaturization results in much reduced sample consumption, increased sensitivity (for same amount injected), reduced operating costs (less mobile phase and disposal), and feasibility of parallel operation. Most of these advantages have been demonstrated in a research setting; however, commercial cLC systems are just now becoming available.

In CE, samples are separated by applying an electric field along a capillary tube, typically 25-75-p.m inner diameter and 10-100 cm long, filled with buffer. The extremely narrow bore of the capillaries allows rapid heat dissipation, because of the high surface area/volume ratio. Because the limit to the electric field applied is typically set by heat buildup from current flow, the capillary arrangement allows electric fields (100-1000 V/cm) that are much higher than those observed in conventional gel electrophoresis (10-30 V/cm). As the resolving power and speed of an electrophoretic separation are directly dependent upon the field applied, CE allows for much faster and higher resolution separations than gel electrophoresis.[2,3] In addition, the extremely small inner diameter capillaries require only nanoliter volume samples, thus reducing sample consumption. In CE, samples are typically injected onto one end of the capillary and then detected near the opposite end. Electroosmotic flow (a type of flow generated in tubes with voltage applied) causes most analytes, regardless of charge, to migrate in the same direction—allowing detection at a single point. Analyte molecules are separated according to their electropho-retic mobility, which is a function of their charge and hydrodynamic radius in solution.

Coupling of liquid separation with MS is relatively straightforward when using continuous flow ionization methods such as ESI. Therefore, ESI has been the method of choice for coupling to modern separation methods, and many proteomic methods have been developed around this combination. MALDI requires samples to be crystallized with matrix, and it is conventionally a vacuum ionization technique. Therefore the coupling of liquid separation to MALDI-MS is more difficult, resulting in a longer development time. Most systems utilize off-line coupling, meaning that the sample is stored on a surface (target plate) that is later introduced to the MS. While off-line methods are seemingly cumbersome, they can present significant advantages such as: 1) independent optimization of separation and MS conditions, and 2) saving the sample after analysis and allowing re-analysis or other experiments such as protein digest to be performed later.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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