Highly Multiplex Targeted Proteomics Enabled by Real-Time Chromatographic Alignment

Targeted mass spectrometry methods produce high-quality quantitative data in terms of limits of detection and dynamic range, at the cost of a substantial compromise in throughput compared to methods such as data independent and data dependent acquisition. The logistical and experimental issues inherent to maintaining assays of even several hundred targets are significant. Prominent among these issues is the drift in analyte retention time as liquid chromatography (LC) columns wear, forcing targeted scheduling windows to be much larger than LC peak widths. If these problems could be solved, proteomics assays would be capable of targeting thousands of peptides in an hour-long experiment, enabling large cohort studies to be performed without sacrificing sensitivity and specificity. We describe a solution in the form of a new method for real-time chromatographic alignment and demonstrate its application to a 56 min LC-gradient HeLa digest assay with 1489 targets. The method is based on the periodic acquisition of untargeted survey scans in a reference experiment and alignment to those scans during subsequent experiments. We describe how the method enables narrower scheduled retention time windows to be used. The narrower scheduling windows enables more targets to be included in the assay or proportionally more time to be allocated to each target, improving the sensitivity. Finally, we point out how the procedure could be improved and how much additional target multiplexing could be gained in the future.