Implementation of MS1 based real–time retention time adjustment on a hybrid Orbitrap mass spectrometer for directed and targeted proteomics

Targeted proteomics studies with large cohorts often suffer from retention time instabilities caused by column aging, especially when low flow HPLC is utilized. To compensate for this, the time windows for scheduling the targets are chosen to be larger, which in turn reduces the number of meaningfully measurable targets.

Real-time correction of retention times provides the possibility to get reproducible identification and accurate quantification of many peptide targets during a LC run regardless of column aging effects which usually result in instabilities of the retention times in large cohorts. Several solutions have already been developed and implemented to tackle this drawback. For example, Exploris Orbitrap mass spectrometers have implemented a landmark-based method that uses isotopically labelled peptides. Although this method is simple and effective, it has the disadvantage that the retention time is corrected at relatively long intervals (distances between the eluting peptides), measuring time is "lost" due to the monitoring of the standards and the costs for the synthetic peptides. A robust method without the need of spiking synthetic standards has been presented by Remes et al.. This method utilizes cross-correlations at MS1 level between reference run and current runs for calculating an estimated RT shift and applies this correction in real-time to the scheduled target list. Here we show the implementation on a modified Orbitrap Exploris 480 mass spectrometer.

For initial assessment we tested different input variables of the algorithm to find the optimal balance between processing speed and correction performance. Additionally, we present results of a targeted long-term study with >150 samples and more than 800 targets as well as a data directed study with more than 5000 targets.

Hela cell digests in different concentrations are used to mimic complex samples. In a first set of experiments, slightly different gradient slopes were used to mimic retention time instabilities and to assess the correction performance. For the long cohort study, replicate injections of HELA digest were used in combination with a nLC column.

Preliminary experiments with 5-10 replicates and retrospective offline processing show a robust retention time correction on replicates of HELA cell lysate samples with differing gradient slopes.

Setting the retention time window to 30s to 45s for a scheduled targeted experiment >95% of the targets (~800) are covered. In data-directed experiments, the window size can be reduced to 8-10 sec, as quantification is performed at MS1 level or via TMT. In this case, up to 95% of a list of 5000 peptides can be reproducibly found across the replicates. Short processing times for cross-correlation calculations and fast data transfer times make real-time correction possible within one scan cycle, thereby delivering superior performance compared to real-time correction based on isotopically labeled standard peptides.