Fuzzier Logic
At the heart of Systems Biology is a vast hunger for measurements. mRNA abundance, metabolite concentration, reactions rates, degradation rates, protein abundance. This last measurement has long been problematic for researchers, mass spectrometers get increasingly accurate and powerful, but are still hindered by the simple fact that observed signal intensity does not necessarily correlate directly with the abundance of that peptide in the sample. Factors such as peptide ionisation efficiencies, dominant neighbour effects, and missing observations all give rise to erroneous estimates of peptide quantities. Until recently, the best way to get close to measures of protein abundance was to use a peptide tagging methodology, but these are typically expensive, and provide only relative quantification (useful for expression proteomics studies, less useful if you need to know the absolute levels of a protein for a Systems Biology study).
Recently, a three step method has been proposed for determining the absolute quantities of proteins in the cell, on a proteome scale. Step one is isoelectric focussing of tryptic digests of whole cell extracts. Step two, calculating the absolute abundance of a small group of proteins by Selective Reaction Monitoring (SRM). SRM uses spike in, isotopically labelled peptides of known concentration as references to calculate the actual abundance of peptides of interest. Finally, step three uses these abundances as reference points to calculate the abundance of all proteins in the sample, using the median intensities from the 3 most intense peptides for each protein.
Leptospira interrogans (Wikimedia Commons)
Using this methodology, the abundances of >50% of the proteome of a human parasite (Leptospira interrogans) have been determined to an accuracy of ~2-fold. These abundance measurements were confirmed by almost literally counting the number of flagellar proteins present in a cell by cryo-electron tomography.
Although current hardware probably limits this technique to a few thousand proteins, that is still a big step forward on what was previously possible. If whole proteome scale absolute abundance measurements become an achievable reality, maybe proteomics can finally take on microarrays as the dominant technique in the post genomics world.
Malmström, J., Beck, M., Schmidt, A., Lange, V., Deutsch, E., & Aebersold, R. (2009). Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans Nature, 460 (7256), 762-765 DOI: 10.1038/nature08184
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