Microscale Solution Isoelectrofocusing

Abstract

Proteomics is a relatively new research discipline that deals with systematic, large-scale analyses of proteins in biological systems (1,2). Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is currently the most commonly used method for quantitatively comparing changes of protein profiles (proteomes), despite a number of limitations, as described below. The basic method utilizes isoelectrofocusing (IEF) in a polyacrylamide gel containing either soluble ampholytes (3–5) or immobilines (6) under denaturing conditions, followed by a second dimension on an orthogonal sodium dodecyl sulfate (SDS) gel. Unfortunately, existing 2-D PAGE methods have inadequate resolution and insufficient dynamic range when used for separating complex proteomes (7,8). A typical “full-size” 2-D gel (approx 18×20 cm) can resolve only approx 1500 protein spots from a cell extract using high-sensitivity stains, whereas about 10,000 genes are typically expressed at one time in a single mammalian cell (9). However, the total number of protein components in higher eukaryotic cells greatly exceeds the number of expressed genes as a result of mRNA alternative splicing and posttranslational modifications (10). Hence, it is highly likely that at least 20,000–50,000 unique protein components comprise typical proteomes from individual mammalian cell types, and the total unique protein species represented in a single tissue probably exceed 100,000. In addition, the quantitative dynamic range (i.e., the difference between the least and the most abundant proteins) in mammalian cells is about 5 to 6 orders of magnitude, whereas the dynamic range in serum is at least 10 orders of magnitude (11). In contrast,