Large-scale, targeted, peptide quantification of 804 peptides with high reproducibility, using Zeno MS/MS 

Using a 20-minute microflow gradient with a Scheduled MRMHR workflow on the ZenoTOF 7600 system

Christie Hunter


A highly multiplex targeted peptide quantification assay has been developed to explore the quantitative capability of Zeno MS/MS on the ZenoTOF 7600 system. Using a mixture of 804 heavy labeled synthetic peptides dosed into plasma, the increase in peptide sensitivity due to Zeno MS/MS was found to be 5.6 fold.  This high sensitivity allowed all 804 peptides to be analyzed in a single time-scheduled MRMHR acquisition using a 20 min gradient, with Zeno MS/MS accumulation times down to 10 msec. Excellent reproducibility was observed with a median CV of 6.1% across all 804 peptides. Concentration curves were also generated to explore the sensitivity in digested plasma, the median LLOQ for the peptides was found to be 207 amol on column.



There are many powerful workflows available for proteomics research on today’s mass spectrometry systems, depending on project goals. They cover a wide range that includes fully untargeted data dependent acquisition approaches for protein identification, comprehensive data independent acquisition strategies for large scale quantification, and also fully targeted quantitative assays for the highest specificity and sensitivity. This last class of assay has been typically performed on triple quadrupole or QTRAP systems because of their very high sensitivity and speed.

The ZenoTOF 7600 system is a QTOF system that can collect high-resolution, high mass accuracy, full-scan MS and MS/MS data. With Zeno trap technology, the system also demonstrates very high sensitivity MS/MS data. The Zeno trap provides significant increases in MS/MS signal: ~5-fold increase for the higher m/z fragment ions that are typically monitored for peptides (Figure 1). Here, a large-scale targeted assay for peptides in human plasma was developed to explore the quantitative capability of Zeno MS/MS on the ZenoTOF 7600 system. Using the PQ500 kit (Biognosys), an MRMHR assay for 804 peptides was run in human plasma, and the reproducibility and sensitivity of the assay were characterized.

Figure 1. Significant gains in peptide area with Zeno trap activated. (Left) Example data of the sensitivity increases observed when the Zeno trap is activated are shown for ANT3.PFLVFIR, with a ~6x gain in peak area. (Right) A summary of the observed sensitivity gains for all 804 peptides is shown, plotted according to precursor mass. The average gain is 5.6-fold.

Key features of the ZenoTOF 7600 system for peptide quantification

  • The ZenoTOF 7600 system delivers a 4 to 20-fold gain in MS/MS sensitivity across the entire m/z range, using the Zeno trap technology1
  • Zeno MS/MS acquired at 10 msec accumulation time with ≥30000 resolution and high mass accuracy
  • For peptide quantification, peak area gains of ~5.6-fold are typical using Zeno MS/MS
  • Excellent quantitative reproducibility and sensitivity was observed for 804 peptides in human plasma, in a single acquisition method, with a 20 min microflow gradient
  • Microflow chromatography and the OptiFlow ion source enable fast gradients with excellent retention time reproducibility for large-scale, time-scheduled, targeted assays