Focus on Phosphoproteins
Mark McDowall, Ph.D., strategic development manager, MS, and James Langridge, Ph.D., senior manager proteomics business, of Waters (www.waters.com), say the characterization of serum and plasma is particularly fraught with peril given the vast span (a factor of 1011) over which protein concentrations range.
“We’ve looked at a number of depletion strategies,” said Dr. Langridge, “most are quite successful in removing the most abundant, targeted proteins, but it doesn’t get you too much further into the serum proteome; depletion is not a perfect solution. Therefore, we approach the problem from the other direction, developing enrichment strategies for the less abundant proteins that are the targets of your investigations.”
Drs. Langridge and McDowall developed a kit based on their investigations that has just been launched by Waters for the capture of phosphopeptides from biological samples. “If you look in the literature, there are a number of groups doing peptide analysis of serum proteins in which the results are biased toward the high-abundance proteins,” states Dr. McDowall. “You sample the same proteins over and over again, so the peptides you detect are overrepresented, resulting in an inability to detect peptides of interest—a situation we refer to as undersampling.”
As they explained, the classic separation of complex protein mixtures in large proteomic studies was most commonly carried out using 2D-PAGE. But in really complex protein mixtures, such as a tumor cell lysate, there maybe several thousand individual proteins and 2-D gel analysis would be overwhelmed due to the complexity and dynamic range.
Waters introduced the Protein Expression System, which substitutes microcapillary liquid chromatography for 2-D gel separation, with a direct connection to the mass spectrometer. This allows identification of the peptide ions with a high degree of quantitative and qualitative accuracy, according to the company. Protein identification is then achieved via database search using the exact mass of the tryptic peptide and fragment ion information.
The Waters Protein Expression System is also designed to perform as a parallel mass analyzer, in which the system alternates between low and elevated collision energy. This allows the acquisition of two data functions from a single LC-MS analysis. The spectra within each function can then be processed yielding highly specific mass data, providing more accurate identification of the target peptides. The system also provides simultaneous qualitative and quantitative proteomics within a single run, so the sample can be mined exhaustively in silico, and up/down regulated proteins and biomarkers can be unambiguously identified.
Although effective sample depletion is demanding, in some situations the Waters group has successfully removed overabundant proteins from complex mixtures. Dr. Langridge and his colleagues depleted proteins, including albumin, IgG, anti-trypsin, IgA, transferrin, and haptoglobin, from serum of patients with Gaucher’s disease. They used a commercial multiaffinity removal system column (Agilent) for this purpose. They analyzed samples of serum from patients using the Waters Protein Expression System and were able to demonstrate a number of marker proteins that may be useful in identifying affected individuals.