Fast-tracking proteomics in the mass spec lab
In many diseases, the difference between healthy and sick can be traced to the body’s cells producing too much – or too little – of a certain protein or proteins. Cancerous cells, for instance, can often develop when tumour suppressor proteins such as p53 are misregulated.
Together, the entire set of proteins produced within a cell, tissue or organism at a certain time is called the proteome. Traditionally, researchers have used mass spectrometers operated in the data-dependent acquisition (DDA) mode to quantify proteomic differences between two samples. Such samples might compare cancer cells to normal cells, or infected tissue to uninfected tissue.
But DDA has some drawbacks that make it hard to accurately and reliably quantify such differences. A more sensitive and more accurate method, known as data-independent acquisition, or DIA, is now producing better-quality proteomics data.
With a state-of-the-art mass spectrometer coming in somewhere between half a million to a million dollars, not many labs can afford their own. That’s what makes the Monash Biomedical Proteomics Facility (MBPF) so appealing. As part of the centrally managed Monash Technology Research Platforms, the MPBF offers high-quality equipment and expert knowledge to help researchers from Monash Uni and other institutions Australia-wide and overseas, as well as commercial clients.
Director of the MBPF, Dr Ralf Schittenhelm, says that DIA mass spectrometry – which the facility was one of the first to implement in Australia – has a major advantage over the traditional method, apart from its accuracy and sensitivity.
“You can globally quantify each and every protein inside a cell: no other technique can do this,” he says. “However, you do have to know which proteins you are looking for. So to analyse the DIA data, you need a library, or a catalogue, of proteins you are interested in.”
A protein library is generated by fractionating and analysing samples supplied by the researcher many times, to ensure that every protein in the cell has been ‘seen’ on the mass spectrometer.
“Once you have the library, you run the DIA experiment on the MS, and screen the data for the proteins present in the library,” explains Dr Schittenhelm.
A computer program compares the peaks on the DIA trace of each sample with those of the library. Just like a scientific game of bingo, when two peaks match, the program ‘calls’ the matching protein.
The folks at the MBPF supply the researchers with a spreadsheet of the results that show which proteins are either upregulated or downregulated, say, in a cancer cell sample compared with a normal cell sample. The researcher then analyses the data and can use the information to discover new biomarkers, identify potential drug targets, and so on.
Dr Schittenhelm says that another great advantage of the DIA system is that once a comprehensive library has been established, it can be used over and over again.
“Say that a few months down the track, you have identified 10 new proteins in your research; you can then ‘reinterrogate’ the same DIA data, and don’t have to run the mass spec again.”
The outcome? A great saving of time and money, which can be directed more effectively to life-saving research.