Over 40% more proteins identified using Zeno MS/MS  

Using the ZenoTOF 7600 system, with the OptiFlow ion source and OneOmics suite in the cloud

Alexandra Antonoplis1, Bradley Schneider2, Christie Hunter1
1SCIEX, USA, 2SCIEX, Canada

Abstract

Proteomics samples are some of the most complex samples analyzed by mass spectrometry, therefore the ability to collect high quality MS/MS at very high acquisition rates is key to achieving high numbers of peptide and protein identifications.  The ZenoTOF 7600 system delivers a ~5-fold gain in MS/MS sensitivity when analyzing peptides, so this study investigates the impact of this large sensitivity gain on identification rates. Four different gradient lengths were tested across a range of sample loadings and a series of acquisition parameters were optimized. Over 40% improvements in the number of protein IDs were observed relative to previous state-of-the-art technology for identical sample loadings.

RUO-MKT-02-13174-A_f0

Introduction

Proteomics samples are some of the most complex samples encountered by the mass spectrometry community. With estimates of over a million different proteins and proteoforms existing in the human proteome,1 understanding which proteins are involved in a given biological function or process can be incredibly challenging. Although any one sample alone would never contain all of these proteins, samples can often contain many thousands of different proteins that can span a concentration range of over 10 orders of magnitude.2 The challenge for discovery proteomics techniques is to try to analyze as broadly and as deeply as possible to detect as many of the proteins and proteoforms present so that biological insights can be gained.

Sensitivity (particularly, MS/MS sensitivity) is a key performance specification for proteomics experiments. High sensitivity MS/MS leads to the broad and deep coverage for protein identification (ID). The ZenoTOF 7600 system uses Zeno trap technology to increase duty cycle to ≥90% across the entire fragment ion mass range, resulting in gains of 4-25 fold in MS/MS mode.3 These gains alone would enable large improvements in proteomics discovery experiments.

The data acquisition strategy is another key factor that can influence proteomics experiments. One of the main acquisition workflows for discovery proteomics is data dependent acquisition (DDA), also known as information dependent acquisition (IDA). During data acquisition, the highest ranking ions that pass certain criteria in each cycle are sent for MS/MS analysis. With many factors to consider, an important consideration is how to specify the selection criteria in order to maximize the number of unique, high confidence peptide and protein IDs. 

In the current study, the sensitivity gains using the Zeno trap technology were evaluated for the impact on protein ID results. Additionally, Zeno IDA acquisition parameters were optimized for 4 different microflow gradient lengths using a design of experiments (DOE) approach. 

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Figure 1. Increase in peptide identifications with the ZenoTOF 7600 system. Peptide Identifications at 1% global false discovery rate (FDR) are plotted in blue for the ZenoTOF system and compared to the TripleTOF 6600 system (red). A 10 min gradient length was used and the TripleTOF system values are an average from three different instruments.4

Key features of the ZenoTOF 7600 system for protein identification

  • The ZenoTOF 7600 system delivers a 4 to 25-fold gain in MS/MS sensitivity leading to many more high confidence peptide and protein IDs
  • Optimized IDA workflows using DOE were determined for a range of microflow gradient lengths by comparing the number of peptide and protein IDs at 1% global false discovery rate
  • Microflow chromatography and the OptiFlow source provide both robustness and ease-of-use for reliable low-flow liquid chromatography
  • Cloud-based ProteinPilot app in the OneOmics suite enables fast and seamless data processing with many features for deriving biological insights

 

Methods

Sample preparation: A digested K562 (human immortalized myelogenous leukemia) cell lysate was used for analysis in all experiments (SWATH Performance Kit - P/N 5045757). The sample was dissolved in water with 0.1% formic acid. Protein loads ranging from 25 ng to 2 µg were used in experiments.  

Chromatography: A Waters ACQUITY UPLC M-class system was used in a trap-elute workflow. A Phenomenex Kinetex 2.6 µm XB-C18 100 Å, 150 x 0.3 mm LC column was used (P/N 00F-4496-AC) with a Phenomenex micro trap (P/N 05N-4252-AC). The column temperature was 30 ºC. Mobile phase A was water with 0.1% formic acid. Mobile phase B was acetonitrile with 0.1% formic acid. Samples were trapped at 10 µL/min for 3 min at 100% mobile phase A. Four linear gradients (5, 10, 20, and 45 min) were tested using a flow rate of 5 µL/min with the following parameters: 5 and 10 min gradients: 3-35% B, 20 min gradient: 3-32% B, 45 min gradient: 3-30% B. 

Mass spectrometry: A SCIEX ZenoTOF 7600 system equipped with the OptiFlow source was used with both the microflow probe and ESI calibration probe.5,8 Source conditions were as follows: CUR, 30 psi; GS1, 10 psi; GS2, 25 psi; IS, 5000 V; TEM, 200 ºC. IDA acquisition parameters that were implemented for all experiments included a TOF MS accumulation time of 100 msec and dynamic collision energy. Only precursors with charge states in the range 2-5 with intensities greater than 100 cps were selected for fragmentation.

As part of the design of experiments (DOE), three acquisition variables were tested: the number of different precursor ions selected for MS/MS for each cycle, the accumulation time for each MS/MS spectrum, and the exclusion time before precursor ions with the same m/z would be reconsidered for MS/MS analysis.

Data processing: Cloud-based data processing was used with the ProteinPilot app in the OneOmics suite.6 All searches were performed as thorough searches, with biological modifications included. Only the resulting proteins and peptides identified at <1% global FDR from fit were used in data analysis. Protein and peptide identification results were visualized and compared in data dashboards in the ProteinPilot app and the FDR (false discovery rate) Comparison Template.7

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Figure 2. Surface plot showing optimization of Zeno IDA parameters. The DOE surface plot enables visualization of the optimal MS/MS and exclusion time parameters for a 10 min gradient length. Quantum XL was used for construction of all models, with a 3 level full factorial design, including 3 factors and 3 replicates. The predictive accuracy of the models was verified at the midpoints for the 3 factors.

Finding optimal IDA parameters

In order to optimize the acquisition parameters for IDA, a DOE approach was used, enabling the construction of models to identify optimal parameters. This approach contrasts with a One Factor at a Time (OFAT) approach in that all parameters can be efficiently explored at the same time, instead of varying one parameter at a time and examining the impact on the response.   For each condition, the number of MS/MS, the accumulation time and the exclusion time were varied, with three values tested for each. A full factorial design for modeling was used with randomization in sample batches in order to generate data using all possible combinations of factors.

Figure 2 shows an example of a three dimensional surface plot generated from the data acquired during the DOE optimization procedures for a 10 min gradient length. From the shape of the surface, the optimal settings can be determined as well as the size of the sweet spot for optimization. The plot makes it very easy to see that the optimal settings exist somewhere around 45 MS/MS per cycle and an exclusion time of around 4 seconds when running a 10 min gradient. The shape of the 3D surface shows that the number of MS/MS per cycle has a large effect on results with lower numbers of MS/MS and shorter exclusion times resulting in fewer peptide IDs overall.

The other plots indicated the best accumulation time was a very fast acquisition rate of 10 msec per MS/MS (data not shown). The DOE results also included a Pareto analysis that indicated the most important setting to consider was the accumulation time for each MS/MS. The ability to acquire MS/MS at extremely fast acquisition rates (down to 5 msec per spectrum) while maintaining high spectral resolution (>30000 for the higher m/z fragments) is a key advantage of the ZenoTOF 7600 system.

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Figure 3. FDR Comparisons Template showing optimization of Zeno IDA parameters at other loadings. The settings identified during the DOE were used as starting points, with additional settings tested to determine optimal settings for other loading amounts. In this example, optimal settings were determined for a 25 ng sample load with a 10 min gradient. Diamonds = proteins at 1% FDR. Circles = peptides at 1% FDR. Red = 45 MS/MS. Blue = 60 MS/MS. Filled diamonds and circles are the averages for proteins and peptides, respectively. 

After the DOE experiments provided the best settings for specific sample loadings, further optimization was performed at a variety of other sample loadings for 10 and 45 min gradient lengths. Results were analyzed using the FDR Comparison Template provided in combination with the ProteinPilot app, which facilitates generation of data visuals from protein ID results. In Figure 3, the number of peptide IDs (circles) and protein IDs (diamonds) are plotted as a function of the accumulation time for a 25 ng sample load injected 3x for each experiment using the 10 min gradient. As shown for this data set, the highest numbers of IDs are obtained with slightly longer accumulation times of 20 msec and 60 MS/MS per cycle. 

When all experiments were completed, a summary table was compiled of the optimal IDA settings as a function of gradient length (Table 1).  As shown for a sample load of 200 ng, the accumulation time optimized at higher values for longer gradient lengths, with MS/MS and exclusion time parameters optimizing in a similar range for all tested gradients. 

Table 1. Settings used for IDA acquisition with 200 ng K562.

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Sensitivity gains using Zeno MS/MS

Orthogonal injection QTOF instruments typically achieve only 5-25% duty cycle for injection of ions into the TOF region.3 This is largely due to the asynchronous nature of coupling of a continuous ion beam source (electrospray) to a mass analyzer that operates under pulsed conditions (orthogonal TOF). The ZenoTOF 7600 system uses Zeno trap technology to increase the duty cycle to ≥90% across the entire mass range. This results in gains of 4-25 fold in MS/MS sensitivity.3 These duty cycle enhancements are accomplished by trapping ions from the collision cell and gating them into the TOF accelerator region such that all ions arrive at the same time and in a more condensed packet.

Figure 4 shows the impact of activating the Zeno trap on the MS/MS signal for a 400 ng injection of K562 cell lysate and 10 minute LC gradient. Optimum IDA settings were used for this gradient length with 45 MS/MS per cycle, 10 msec accumulation time and 5 second exclusion time. The pink trace shows the MS/MS total ion chromatogram (TIC) obtained with the Zeno trap on while the blue trace shows the Zeno trap off. When using the Zeno trap, a large increase in TIC height is observed. Additionally, quality scores for MS/MS spectra increased greatly as well. Using a score threshold of 95,  87% of scores for the data with the Zeno trap on exceeded this threshold, while only 55% of spectra exceeded this score for the data with the Zeno trap off. The resulting increases in protein and peptide are summarized in Table 2 and Figure 5.

Figure 5 shows the comparison data for Zeno trap on and off for injection of 200 ng K562 cell digest for all gradients tested. Here, the peptide IDs are plotted as a function of the gradient length. While specific gains vary as a function of gradient length, gains of more than 55% are observed for longer gradients as shown in Figure 5 and Table 2.

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Figure 4. Impact of using Zeno trap for IDA. The MS/MS total ion chromatograms (TIC) for IDA data acquisition are shown for a 400 ng sample load and a 10-minute gradient. Using the Zeno trap (pink) results in a large gain in signal versus having it off (blue). The MS/MS total ion chromatograms (TIC) for IDA data acquisition are shown for a 400 ng sample load and a 10-minute gradient. Using the Zeno trap (pink) results in a large gain in signal versus having it off (blue).

Table 2. Increase in peptide identifications for the Zeno trap for injection of 200 ng K562 cell digest.

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Figure 5.  Increase in peptide identifications with the Zeno trap activated. Peptide IDs at 1% global FDR are plotted as a function of gradient length for 200 ng K562 cell digest (see Table 1 for acquisition methods). The Zeno trap greatly increases the number of IDs at all gradient lengths (see Table 2).

More peptides and proteins identified

A comparison study was performed for the ZenoTOF 7600 system versus the TripleTOF 6600 system to better understand the gains achieved from the new instrument as a whole. Both short and long gradient conditions were tested as well as many different protein loading amounts with the aim of covering a range of typical laboratory scenarios.  

Figure 6 shows the number of proteins identified for replicate protein loadings using a 10-minute gradient. When looking at the low end of the curve, loadings of only 100 ng produce more peptide and protein IDs than loading 5x more (500 ng) on the TripleTOF 6600 system. Table 3 summarizes the large percentage of gains for 1000 ng protein loadings as observed using the ZenoTOF 7600 system.

Figure 7 shows the same experiments, but for a 45-minute gradient, with replicate loadings for the ZenoTOF 7600 system plotted. Again, the ZenoTOF 7600 system produces many more peptide and protein IDs, and the percentage of gains for 1000 ng are summarized in Table 3. 

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Figure 6. Instrument comparison for a 10 minute gradient. More peptides (top) and proteins (bottom) are identified for the ZenoTOF 7600 system (blue) versus the TripleTOF 6600 system (red). The TripleTOF system reference values are an average from three different instruments.4

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Figure 7.  Instrument comparison for a 45 minute gradient. Comparison of identified peptides (top) and proteins (bottom) between the ZenoTOF 7600 System (blue) and the TripleTOF 6600 System (red). Prior data collected on the TripleTOF 6600 system at 1 µg load (average of three instruments) is visualized for reference.4

Table 3. Percentage of gains in peptide and protein IDs for the ZenoTOF 7600 system versus the TripleTOF 6600 system at a 1000 ng sample load.

Conclusions

The ZenoTOF 7600 system provides significant gains in peptide and protein identifications for proteomics experiments. These gains come from a combination of the unique Zeno trap technology and the optimization of acquisition parameters for a series of different workflow scenarios.

  • Up to ~45% improvements in the number of protein IDs and up to a 145% increase in the number of peptide IDs versus previous state-of-the-art technology for identical sample loadings
  • Alternatively, the increased sensitivity can be used to provide very good protein identification results using 5x lower sample loading, when sample amounts are limiting
  • The large increase in the number of IDs can be used to improve the quality of libraries generated for SWATH data processing
  • As a complete solution using microflow chromatography, the OptiFlow source and cloud-based ProteinPilot App enable a robust, sensitive and streamlined workflow from acquisition through results generation.

 

References

  1. Ponomarenko, E.A., et. al. (2016) The Size of the Human Proteome: The Width and Depth, Int. J. Anal. Chem., Vol. 2016, ID 7436849. 
  2. Anderson, N.L., et al. (2004) The human plasma proteome, Mol. Cell. Proteom., 3 (4), 311–326.
  3. Qualitative flexibility combined with quantitative power. SCIEX technical note, RUO-MKT-02-13053-A.
  4. Fast protein identification experiments with microflow – Up to 100 Samples Per Day. SCIEX technical note, RUO-MKT-02-8432-A.
  5. Single source solution for low flow chromatography. SCIEX technical note, RUO-MKT-02-9701-A.
  6. OneOmics overview. SCIEX technical note, RUO-MKT-6569-B.
  7. FDR comparison template for easy comparison of FDR results from ProteinPilot software results. SCIEX community post, RUO-MKT-18-10968-A.
  8. Automatic calibration in minutes for microflow LC  with the OptiFlow Source on ZenoTOF 7600 system, SCIEX community post, RUO-MKT-18-13325-A.

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