Methods

Samples and reagents: Universal Proteomics Standard (UPS) was purchased from Sigma-Aldrich. Rat plasma (Sprague Dawley, K2 EDTA) was purchased from BioIVT.

Sample preparation: The calibration curve was prepared by spiking digested UPS into rat plasma digest followed by serial dilution.

Samples were denatured by incubating with N-octyl-glucoside (OGS), followed by reduction with dithiothreitol (DTT) and alkylation with iodoacetamide (IAM). A trypsin/Lys-C digestion was performed at 37 ºC overnight, with an enzyme-protein ratio of 1:25. Formic acid was spiked into the samples to abort digestion. The samples were centrifuged at a speed of 12,000 g and the supernatant was injected for LC-MS analysis. 

As a note, proteins used for this study had limited starting concentrations. Therefore, the final LDRs were narrow for the peptides analyzed.

Chromatography: An ExionLC system was used for analyte separation. A volume of 20 µL was injected for analysis. Mobile phase A consisted of water with 0.1% FA in water, while organic phase B was composed of 0.1% FA in acetonitrile. For analyte separation, the operating flow rate was set to 0.5 mL/min using a Phenomenex Kinetex C18 column (3 x 50 mm, 2.6 µm, 100 Å). The column oven temperature was set to 40 ºC. Chromatographic conditions for analyte separation are shown in Table 1.

Mass Spectrometry: Samples were analyzed in triplicate. Method details such as source and gas parameters and MS conditions are summarized in Table 2. Sample analysis was performed using scheduled Zeno MRM^HR on the ZenoTOF 7600 system. The ZenoTOF 7600 provides a scan speed of 133 Hz.

Data processing: MRM data were processed using SCIEX OS 2.0 software. Integration was performed using the MQ4 algorithm. Linear regression with 1/x weighting was used for quantification of all peptides. The XIC peak width was set to 0.05 Da.

Greater sensitivity with Zeno trap
 

With traditional time-of-flight MS/MS, fragment ions arriving from the collision cell are often lost in transmission between TOF pulses due to differences in velocity. As a result, for standard time-of-flight MS/MS, duty cycle range is approximately between 5-25%. A decrease in sensitivity occurs as a consequence of loss in ion transmission. The Zeno trap ensures greater ion transmission by controlling the ion beam from the collision cell into the TOF accelerator (Figure 2). Ions exit the Zeno trap based on potential energy.

The Zeno trap enabled significant improvements in MS/MS sensitivity. On average, 5-fold improvement in LLOQ was achieved using Zeno MRM^HR in comparison with standard MRM^HR (Figure 1). Out of 48 peptides measured, 75% of peptides showed ≥3-fold improvement in LLOQ.

Overall, XICs comparing LLOQs with MRM^HR and Zeno MRM^HR show significantly lower LLOQs were achieved with the Zeno trap (Figure 3). Strong linearity was achieved for all peptides analyzed (Figure 4). Accuracy at the LLOQ was within 80%- 120%, while for all other non-zero calibrators, accuracy was within 85%-115% of the nominal concentration. Overall, precision was <15%, demonstrating high reproducibility (Table 3).

Summation of multiple fragment ions enhances sensitivity

The accessibility of TOF MS/MS data can be advantageous as post-acquisition data decisions can be made on which measured fragments can be utilized for MRM^HR. For MRM^HR, quantification can be performed using single fragment ion or by summing multiple dominant fragment ions. When multiple, high-abundant, fragment ions are generated from the target peptide, the sum of XICs can further enhance the assay sensitivity.

As shown in Figure 5, summing of multiple dominant fragment ions can achieve up to a 3-fold improvement in LLOQ. Strong linearity was achieved for quantification with single fragment ions and summed multiple fragment ions (Figure 6). Accuracy at the LLOQ was within 80%-120%, while for all other non-zero calibrators, accuracy was within 85%-115% of the nominal concentration. Overall, precision was <14%, demonstrating high reproducibility (Table 4). 

As discussed earlier, the Zeno trap provides added sensitivity enhancement through improvements in duty cycle. The cumulative gain from the use of the Zeno trap and summation of highly abundant fragment ions enhances overall assay sensitivity (Figure 7)

Conclusions
 

• An average of 5-fold in LLOQ improvement was achieved for peptide quantification in this sample set using the Zeno trap, which enhances the duty cycle through the accumulation of ions during each TOF pulse 
  
  
• Improved LLOQs were reached by summing of multiple highly abundant fragment ions along with the availability of TOF MS/MS data, resulting in a 3-fold enhancement in LLOQ
  
  
• A highly accurate and reproducible quantitative workflow for peptides was demonstrated using the ZenoTOF 7600 system
  

References
 

1. Mike-Qingtao Huang, Zhongping (John) Lin, Naidong Weng (2013). Applications of high-resolution MS in bioanalysis. Bioanalysis 5(10):1269-1276. 
   
   
2. Yuan-Qing Xia, Jim Lau, Timothy Olah, Mohammed Jemal (2011). Targeted quantitative bioanalysis in plasma using liquid chromatography/high‐resolution accurate mass spectrometry: an evaluation of global selectivity as a function of mass resolving power and extraction window, with comparison of centroid and profile modes. Rapid Communications in Mass Spectrometry 25(19):2863-2878.