Ruedi Aebersold and Hui Zhang of the Institute for Systems Biology outline a method in US 2010/0222233 to address two current limitations in protein analysis: the inability of microarrays to reliably quantify protein amount and the inability of mass spectrometry to detect proteins of low abundance. The ISB approach is essentially to use both methods in sequence, with a microarray step preceding a MALDI-MS step. The use of an initial array step to retain peptides of interest (including fragments of low abundance proteins) enables subsequent MS analysis of low abundance proteins, and detection settings can be tuned for specific protein fragments to further enhance sensitivity. As outlined in the patent, the challenge in quantification with microarrays is that target protein and array element pairs will each have different optimal binding conditions, and target proteins will have solubility variations that are also dependent on solution conditions. However, use of heavy isotope peptide standards of known concentration solves this problem: Loss of target peptide during washing steps on the microarray will be accompanied by similar loss of its heavy-isotope peptide-standard counterpart, but the exact starting quantities of the heavy-isotope peptides are known, so the ratios of resulting MS signals (heavy/light) will yield the quantities of target peptides.
The approach outlined in the patent is an advance to the state of the art, but remains limited as a proteomics tool since it only works for previously characterized proteins. Basically, you have to know what you’re looking for already. It’s worth pointing out that the idea of separating incoming molecules prior to mass spectrometry analysis is not new (e.g., GC-MS instruments were in widespread use beginning around 20 years ago). However, the protein chip’s high degree of specificity and high-throughput nature – when used in combination with mass spectrometry – is new.
Copyright © Bruce A. Schiamberg 2010. All rights reserved.