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.