The move towards miniaturization in analytical chemistry has prompted the development of new formats for sample preparation. The micropipette tip format permits the handling of submicrolitre amounts of samples such as biological fluids. Solid-phase extraction (SPE) has been performed with various phases packed, embedded or coated on the walls of the pipette tip. The open flow architecture allows liquid samples to be moved and transferred without undue pressure drop or plugging. Many popular SPE techniques have been demonstrated including reversed-phase, ion exchange, hydrophobic interaction, hydrophilic interaction, immobilized metal affinity and affinity chromatography. Aside from SPE, micropipette tips have also been used to miniaturize dialysis and enzyme digestion. This month's instalment of "Sample Preparation Perspectives" reviews the latest technologies in micropipette tip sample preparation used in the study of genomics, proteomics and metabolomics.

In the past 10 years, the structural determination of proteins by the use of mass spectrometry (MS) has created new opportunities and challenges in analytical chemistry and biochemistry. The newly developed techniques provide the potential to map human proteins similar to the way a first blueprint of the human genome was announced a few years ago. Yet, the Human Proteome Project has more challenges than the Genome Project because of the complexity of protein structure and post-translational modifications, which often consist of dynamic changes. MS, by providing protein detection sensitivity at levels of 1 amol or lower, represents a breakthrough technology for analysing the structures of proteins in a single cell.

One of the primary challenges of proteomics is to determine the structures of biologically functional molecules from among a pool of millions of proteins, each with its own dynamic structure and unique post-translational modifications. While MS provides detection sensitivity, proteins still need to be purified before analysis. Some scientists assert that the purification of proteins before MS analysis is unnecessary because, in their view, a representative fragment of a protein can be identified by MS and then matched against a computer database of protein sequences. Yet, the current limitation of this method is that it might miss the true functions of proteins based upon their post-translational modifications. On the other hand, the purification of proteins before MS analysis can also result in the loss of, or changes to, the post-translational modifications of proteins and thus represents many of the same limitations. Another challenge to pre-MS protein purification is obtaining sufficient quantities of the protein of interest.^1,2 For example, in human plasma, 10 proteins represent 90% of protein weight and an additional 200 proteins represent 9% of the remaining weight. The remaining millions of proteins account for less than 1% of the protein weight of plasma.

The first set of analytical tools, developed decades ago, were based upon the isolation of several grams of proteins to determine their structure. Today, we use most of the same techniques: adsorption, partition, ion exchange and affinity chromatography,³ metal chelation, electrophoresis (1-D or 2-D)⁴ and SPE, but we have reduced our scale to micro levels for increased sensitivity. For example, when 2-D-electrophoresis was developed, one could obtain about 2000 spots from a sample. Today, the resolution is not much better; one still obtains about 2000 spots out of a set of millions of proteins. Each spot could contain hundreds of thousands of proteins, to be further analysed.