Simplified and Efficient Quantification of Low-abundance Proteins at Very High Multiplex via Targeted Mass Spectrometry*□S

Liquid chromatography–multiple reaction monitoring mass spectrometry (LC-MRM-MS) of plasma that has been depleted of abundant proteins and fractionated at the peptide level into six to eight fractions is a proven method for quantifying proteins present at low nanogram-per-milliliter levels. A drawback of fraction-MRM is the increased analysis time due to the generation of multiple fractions per biological sample. We now report that the use of heated, long, fused silica columns (>30 cm) packed with 1.9 m of packing material can reduce or eliminate the need for fractionation prior to LC-MRM-MS without a significant loss of sensitivity or precision relative to frac-tion-MRM. We empirically determined the optimal column length, temperature, gradient duration, and sample load for such assays and used these conditions to study de-tection sensitivity and assay precision. In addition to in-creased peak capacity, longer columns packed with smaller beads tolerated a 4- to 6-fold increase in analyte load without a loss of robustness or reproducibility. The longer columns also provided a 4-fold improvement in median limit-of-quantitation values with increased assay precision relative to the standard 12 cm columns packed with 3 m material. Overall, the optimized chromatogra-phy provided an approximately 3-fold increase in analysis throughput with excellent robustness and less than a 2-fold reduction in quantitative sensitivity relative to fraction-MRM. The value of the system for increased multiplexing was demonstrated by the ability to configure an 800-plex MRM-MS assay, run in a single analysis, comprising 2400 transitions with retention time scheduling to monitor 400 unlabeled and heavy labeled peptide pairs. Molecular &amp