45. Optimizing the Peptide Quantitation Workflow from Biomarker Discovery to Validation
Poster: Mon 2:00-3:00PM
Christine A Miller
Agilent Technologies
Christine A Miller, Agilent Technologies
Ning Tang, Agilent Technologies
Keith Waddell, Agilent Technologies
Quantitative proteomics often requires monitoring of hundreds of target peptides in thousands of biological samples. Acquiring MRM transition ion data only during the retention window for each peptide reduces the overall number of ions monitored at any one time and therefore improves sensitivity. Optimizing the collision energy for the selected transitions also improves sensitivity. In this study, standard peptides were optimized, and then these peptides were spiked into human plasma for quantitation. LC/MS analyses were performed on a triple quadrupole (QQQ) or Q-TOF mass spectrometer interfaced to a microfluidic-based nanoflow LC system. Software was used to help design and optimize MRM transitions from the standard peptides. Additional MRM transitions were selected from discovery data (Q-TOF) collected from the complex samples. These transitions were used to examine the impact of the number of dynamic MRMs in a method on quantitation of the target standard peptides.

For the standard peptides, an automated MRM optimization routine was applied that primarily determined the optimal collision energy for selected transitions and results were validated against manual optimization results. The spiked plasma digest was first analyzed using a Q-TOF system for protein identification and retention time determination. The average standard deviation for retention time was less than 0.1 minutes from triplicate runs. Two proteotypic peptides from each selected protein based on the peptides identified from the Q-TOF experiment. The software selected the peptides based on the amino acid requirements (length, absence of Cys and Met etc.), missed cleavages and the uniqueness in the database. For the selected peptides, two MRM transition pairs were selected for each peptide based on intensity. A total of more than 400 MRM transition pairs were generated to be analyzed in a single QQQ run. The same gradient as in the Q-TOF analysis was used with QQQ so the retention time value could be used to trigger MRM acquisition. The quantitation results were measured in 5 replicates with and without retention-time triggering of the MRM transitions. The performance of these two experimental conditions will be reported and the effect of the two methods on the quantitation results of the spiked proteins will be compared.