Everything you need to perform protein quantitation: The starter kit contains peptide standards, sample preparation reagents and buffers, trypsin, a column, data acquisition methods, and processing methods and templates for performing protein quantitation using multiple reaction monitoring (MRM) analysis on an SCIEX Triple Quad or QTRAP System. The peptide standards for each protein are synthesized using isotopically-enriched amino acids (lysine and arginine) to provide the highest quantitative robustness. The LC/MS/MS method allows for a direct measurement of CYP450 protein levels without the use of antibodies, providing savings in both time and expense (Figure 1).
Figure 1: Example MRM data.
Advanced data analysis software. The data generated for CYP450 analysis is processed with MultiQuant Software. The software automatically integrates the MRM data and computes area ratios to the added peptide internal standards for all samples. Processed area ratio results are imported into a Microsoft Excel template for computation of protein level data. Results are plotted in a simple bar chart to compare sample expression levels of each CYP450 isoform to negative control expression levels.
Figure 2: Assay type comparison.
Correlation between enzymatic activity, mRNA expression, and protein expression (as measured by LC/MS/MS) is excellent (Figure 2). Both baseline and inducible levels of the CYP450 isoforms are easily detected by the LC-MRM protein quantitation method.