A Method for Automated Multistep (Multidimensional) Purification Processes for Protein Recovery

MAY 2005 C hromatography is the most powerful separation tool available to protein scientists and plays a central role in proteomics and functional biology. Problem solving in both areas requires purified proteins, but the purification requirements are different. In both types of research the samples are often complex mixtures derived from tissue homogenates, body fluids, or cell cultures. In proteomics, chromatography is primarily used to prepare samples for mass analysis and subsequent identification. A chromatographer in that environment focuses on high-thoughput analysis. The multidimensional aspect of chromatography in proteomics can involve ion exchange, affinity, and size exclusion as the first dimension and desalting by reversed-phase chromatography as the second dimension. Proteins are separated from complex mixtures and desalted before mass spectrometry, an area that has been recently reviewed by Wang and Hanash (1) and Shi and coworkers (2). The importance of high throughput to these strategies is reflected in the development of chip technology for such separations (3). In functional biology, a chromatographer has a different focus. Here it is more important to recover the purified protein than to identify it. Multidimensional chromatography in this area uses desalting as the first dimension and some adsorptive technique (ion exchange, hydrophobic interaction, or affinity) as the second dimension. Multidimensional chromatography is not a new strategy for protein recovery. In the early 1960s, Lawrence and Benjamin used it to study blood proteins (4), and it has since been a consistent strategy for purifying proteins in cases when protein recovery is important (5, 6). Fully automating multidimensional chromatography for protein recovery requires a capital outlay for new instrumentation. However, in the absence of such capital, the prevalence of chromatographs with sophisticated chromatography control software packages makes it easier for many laboratories to automate multidimensional chromatography. We describe here a protocol to configure existing instruments for multidimensional chromatography. Because it does not require new instrumentation, it can be done with a minimal outlay of capital.