A profile-directed biomarker discovery approach involving initial differential expression analysis followed by targeted identification of differentially expressed proteins was used in this study. Using Agilent platforms such as a Multiple Affinity Removal (MARS) column, a 1200 Series HPLC-Chip/MS and a 6520 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS, excellent reproducibility in retention time and intensity measurement, and outstanding accuracy in mass measurement and sensitivity over a wide dynamic range were achieved. Differential analyses using MassHunter Mass Profiler and MassHunter Mass Profiler Professional (MPP) software enabled the comparison between spiked and non-spiked plasma samples. Subsequent targeted MS/MS analysis on a Q-TOF LC/MS followed by a database search using Spectrum Mill helped identify the spiked protein.
The document outlines a study demonstrating the identification of horseradish peroxidase spiked into human plasma using Agilent's 1200 Series HPLC-Chip/MS and 6520 Accurate-Mass Q-TOF LC/MS platforms. The workflow involves protein depletion, enzymatic digestion, and analysis to detect differentially expressed proteins.
The study used varying amounts of spiked protein, specifically 100 femtomole and 10 femtomole.
The retention time deviation among four replicate LC/MS runs is within +4 sec.