Analysing a cell's full complement of proteins can be a fairly time-consuming process. As such, protein scientists are often caught on the horns of a dilemma: how to analyse all of a cell's proteins in a reasonable amount of time but in sufficient detail to identify individual proteins accurately.

This dilemma is especially acute when, as is usually the case, a cell's proteome is analysed using a combination of liquid chromatography (LC) and tandem mass spectrometry (MS). For the enhanced information about a protein that can be obtained by performing repeated analyses using something like an ion trap-Fourier transform mass spectrometer (FTMS) is offset by the need to keep up with the analytes continually eluting from the LC column.

One way around this problem is to digest the protein into its component peptides, which are quicker and easier to analyse with a mass spectrometer than whole proteins. But determining the identity of the parent protein from their component peptides can be far from easy. Indeed, a 2003 study estimated that 13% of all the proteins found in yeast cells using this method were likely to be identified incorrectly.

An alternative way to resolve the dilemma would be a system that could automatically highlight those proteins that are of most potential interest, especially those that appear to be novel, based on an initial, quick analysis by standard LC/MS. These highlighted proteins could then by shunted off for a more detailed analysis by tandem MS, with the system also deciding how many analyses need to be conducted to derive the maximum amount of information and therefore produce an accurate identification.

In this way, detailed, time-consuming analyses could be saved for those proteins that really need them. Furthermore, these detailed analyses would be performed separately from any chromatographic separation, ensuring that the two processes don't interfere with each other.

Well, a team of chemists from the University of Illinois at Urbana-Champaign, led by Neil Kelleher, has now gone a fair way towards producing just such a system. 'Any proteomic study which is ultimately limited by the amount of time available for analysis during chromatographic elution could benefit from this platform, which is virtually all of them,' claims team member Craig Wenger.

As with the ideal system, the automated system developed by Wenger and his colleagues performs a preliminary analysis of a cell's proteome using LC/MS. It then automatically identifies those protein peaks that haven't been spotted before, based on their mass, and oversees a more detailed FTMS analysis of just these proteins.

This dual analysis is achieved by splitting the HPLC eluent in two. The system only sends a very small amount to the LC/MS, while preserving the vast majority (over 99%) for the FTMS analysis.

'This is not the first completely automated system for proteomics by tandem MS, but it is certainly one of the most advanced,' Wenger told separationsNOW. 'It is the first automated system to link online and offline strategies to get the most complete picture of the proteome. The software ensures there is sufficient electrospray signal before collecting every scan of data, and we make data-driven decisions about if it is worthwhile to acquire data on a target and also how many scans should be averaged.'

Using this system, Wenger and his colleagues were able to identify 57 different proteins in the nucleus of a human cell, whereas a more conventional analysis yielded only 16 firm protein identifications. The entire process took 15 hours, but required no human intervention: after being started off, the system could be left alone until it finished.

Wenger and his colleagues are now looking to get their system closer to the ideal by letting it use information about earlier protein matches to help determine whether a detected protein should be analysed in more detail. Those proteins that are already well-characterised would not be strong candidates for such a detailed analysis.

Related links:

• Analytical Chemistry (Article in Press): "Versatile online-offline engine for automated acquisition of high-resolution tandem mass spectra"

Article by Jon Evans

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.