The complexity of biological samples combined with the relatively low sample amounts often available presents a significant barrier to the successful analysis of biological samples. Miniaturization of separation techniques offers one potential methodology for overcoming the complexity/paucity barrier. This is an approach we have developed extensively in combination with mass spectrometric detection, and includes nanoscale packed capillary LC and capillary electrophoresis (CE). A feature common to these techniques is the capability of delivering high concentrations of low level analytes in small volumes to the detector. We have interfaced these separation techniques with a variety of mass spectrometric techniques including fast atom bombardment, electrospray ionization and laser desorption ionization. In addition to packed capillary LC and CE, we have developed methodology for directly determining the mass of analytes affinity-bound to an immobilized affinity column or to an immobilized metal ion affinity column. This has proven to be an essential component of our structural studies. We are also systematically investigating the effect of organic co-solvents on capillary electrophoretic behavior. Use of organic co-solvents can improve separations in two ways: 1) analysis time is changed due to changes in ionic strength and electric field strength which can increase the separation efficiency as compared with aqueous media; and 2) hydrophobic compounds, such as lung surfactant proteins and lipoproteins, can be solubilized and thus made amenable to analysis. We have used these techniques to separate and analyze oligonucleotide digests, apolipoproteins and apolipoprotein digests, complex venoms, and digests of carcinogen-exposed mouse hemoglobin digests. These techniques are wholly integrated in our applications projects.
