Protein Profiling of FFPE Tissue
According to Casey Eitner, CEO of Expression Pathology (www.expressionpathology.com until the Liquid Tissue® MS Protein Prep Kit was launched, there was no way to do MS analysis on formalin-fixed tissue. “This opens up a whole new avenue for protein analysis, biomarker discovery, and protein target discovery.”
This automated protocol solubilizes the entire tissue sample so there is a representation of the total protein content. “It has been optimized to work with 30,000 cells, which isn’t a lot, but it’s the amount you can typically microdissect. This allows users to harvest specific cellular features from a single tissue section and perform detailed proteomic analysis of those cellular features,” notes Eitner.
Liquid Tissue has two main MS applications—to identify differently expressed proteins associated with a specific condition or clinical outcome (e.g., which proteins change as cancer progresses) and once proteins of interest have been identified, using MS with single-reaction monitoring (SRM) or multiple-reaction monitoring (MRM) to quantitate the target protein(s) in the changing clinical circumstances.
“We’re just starting to offer the ability to specifically quantitate changes in protein expression in FFPE using SRM and MRM spectrometry,” says Eitner. “This can be done with labeled peptides or in an unlabeled manner.” Eitner thinks this technology will provide protein-expression analysis alternatives.”
Mass spectrometry is technically challenging, and the sample prep prior to running MS is especially important. “It’s not just about running things through the mass spec, it’s about integration and putting the correct thing into MS and interpreting results properly,” says Eustache Paramithiotis, Ph.D., director of cellular and molecular biology at Caprion Proteomics (www.caprion.com). “We’ve seamlessly integrated all the steps involving sample prep, analysis, and informatics to understand results and made it an industrialized process with QA and QC.”
The resultant CellCarta® proteomics platform allows the comprehensive detection of disease-relevant protein targets and biomarkers across a wider dynamic range of concentrations, according to Dr. Paramithiotis. Sample prep enables isolation of organelles. This provides enhanced proteomic comparisons of normal and diseased cells as well as the biological and functional context for identifying disease protein targets.
Caprion has 11 LC-MS systems capable of processing up to 250 biological samples per week. Samples are first analyzed via LC-MS to detect differentially expressed peptides and then reinjected in to LC-MS-MS to obtain sequence information for those peptides. Analytical tools transform raw LC-MS and LC-MS-MS data into reliable protein-expression information.
“With proteomics, you have to look at the overall picture from beginning to end, not just what MS does. You can have an absolutely precise instrument, but if your study design is poor and your sample prep is variable, then what good is it? Proteomics is not just about giving a list of proteins to the end user, it is also about giving some interpretation of what they mean,” says Dr. Paramithiotis.
Overall, mass spec is being adapted for more diverse applications. Sample prep continues to be a crucial step in MS that must be performed with great accuracy. The technology will continue to advance as the research requires. It remains to be seen, however, how the new data will be used. “There will always be a technological element, better machines, etc., but the technique’s penetration—how well the answers will be utilized—that’s the crux of it,” summarizes Dr. Paramithiotis.