Smith and colleagues outline the new method and how it is used for the high-throughput, quantitative monitoring of relative protein abundance between cell lines exposed to different conditions. To globally label the proteins in test and control populations, the researchers grow the two populations in different media. Furthermore, for every protein in the test cell, there is a corresponding labeled protein made by the control cell. Thus, the control cells offer the ideal internal standard, Smith says. The rest of Smith's sample preparation is similar to that developed by others.
Cells are lysed and digested with trypsin to give peptide fragments.
Peptide fragments that contain a cysteine residue are tagged with an affinity reagent and separated from the other fragments. This simplifies the chromatography and increases the sensitivity to weakly expressed proteins. Whereas other researchers use this tagging step to impart the isotopic label, Smith's group has already labeled the peptides, so the two populations can be combined before sample preparation. This eliminates any variance that might be introduced at this stage. The only limitation to the new method is that it can't be used for unculturable cells or multicelled organisms, which would not necessarily incorporate the label into every cell, Smith notes.