Isobaric labeling techniques TMT have become popular for biomarker discovery due to higher throughput and better precision and accuracy. The next verification step (10-50 patients) is challenging when balancing the target numbers and devoted instrument time. Here, we propose a workflow for plasma proteomics from multiplexed TMT biomarker discovery to rapid and robust verification using capillary flow LC on a novel Orbitrap platform with up to 40Hz scan speed.
Plasma from normal and diabetic patients were depleted, digested, labeled with TMT six-plex reagents, mixed at 1:1 ratio, and fractionated. The fractions were separated in a 120min gradient followed by analysis on an Orbitrap instrument. Data were analyzed by Proteome Discoverer™2.2 software. For targeted analysis, the same depleted, but unlabeled, samples were separated at a flowrate of 2-5ul/min and analyzed using parallel reaction monitoring (PRM). The data were processed by Skyline or Spectronaut software.
The multiplexing capability of TMT labeling significantly saved instrument time and provided possibilities to perform extensive fractionation. Fractionation, combined with the new depletion columns, made the detection of plasma proteins spanning to 5 orders of magnitude accessible.
Over 200 peptide targets, which showed significant difference (>2 fold change) between normal and disease states in the above discovery experiment, were selected for label free targeted quantitation using PRM. Retention time prediction of unlabeled peptides was performed using adjusted hydrophobicity index calculations. LC separation at the capillary flow rate provided high sensitivity and offered improved retention time reproducibility and robustness. The fast scan speed on the new Orbitrap platform greatly facilitated the detection of hundreds of targets in a single experiment. This rapid and robust quantitation method confirmed the biomarker candidates found in isobaric labeled discovery experiment.
The workflows described here enable the biomarker discovery and validation in a highly multiplexed and rapid manner.