Evaluation of Strategies for High Sensitivity Protein Monitoring in Human Plasma using Liquid Chromatography-Multiple Reaction Monitoring/Mass Spectrometry (LC-MRM/MS)

摘要

Performance of LC-MRM/MS for protein profiling in human plasma has been evaluated using a 33KDa concatamer protein (QconCAT) derived from chicken. A BLAST search revealed no peptides utilized in the assay had any identity with human proteins. We were able to utilize a heavy (~(13)C_(6) lysine and arginine) and light version of the QconCAT protein. The assay was developed using a LTQ-Orbitrap XL in CID and HCD mode and transitioned to a TSQ Quantum tandem quadrupole. The CID data most closely matched the MRM transition ratios. Analysis of the QconCAT alone showed detection of 1.5pg on column. We evaluated two analytical strategies for detection - depletion and depletion/fractionation followed by digestion. Method 1 showed detection at the 50ng/mL level (200ng digest loaded on-column). with approximately 50percent loss during sample work-up. CVs for technical replicates increased from 5percent to 23percent going from 5(mu)g/mL to 50ng/mL. Method 2 demonstrated the ability to detect lower than 1ng/mL through analyte enrichment. These methods are universally applicable to any protein.