17
The system maintains high sensitivity across wide dynamic ranges, capturing both abundant and scarce metabolites, which is critical as metabolite concentrations span orders of magnitude [3]. Zamboni observed a six-order-of-magnitude intra- scan dynamic range (10–10 million), enabling improved MS1/MS2 feature detection using DDA and DIA workfl ows, and facilitating aggressive peak-picking to detect and characterise more metabolites than previously possible [3,11].
Ultrafast data-independent acquisition unlocks the complexity of metabolomics
With DDA, they found the number of features detected increased two- to three-fold in a lipidomics experiment compared with the ZenoTOF 7600 system [3,11]. Double the number of precursors were fragmented, and by matching the data against a library of theoretical MS2-fragments, there was approximately a 50% increase in putatively MS2-annotated lipids, without any loss in match quality observed [3,11]. Even in a challenging dilution series, they were able to detect many more features without any tangible loss in matching statistics [3,11]. For instance, at a 1000x dilution, they detected fi ve times more features (see Figure 2) [11]. In a similar metabolomics experiment with DDA, the ZenoTOF 8600 system provided 80% more identifi cations [11]. This translates to being able to detect and identify more of the metabolites, especially the less abundant ones, and with the same confi dence that they had with the more abundant ones [3]. Moreover, the improved sensitivity also meant that their DDA experiments with EAD could be sped up by a factor of three to fi ve [3].
Figure 3: In lipidomics, ZT Scan DIA 2.0 led to 50% more MS2-based identifi cations than DDA (15-minute method), with better matching, with the exception of spectral purity, pointing to an increase in chimeric MS2 spectra. ZT Scan DIA 2.0 was pushed even further, to fragment all precursors in a cycle time of 400 ms, including MS1. In a 2.2-minute LC-MS, Zamboni and his team putatively annotated 1300 lipids by MS2 [11].
Figure 2: To test whether the increased sensitivity also applied to the most challenging analyses, Zamboni and his team analysed a dilution series on a short 2.2-minute reverse phase LC-MS method. At a 1000x dilution, the ZenoTOF 8600 system detected 5 times more features, with no tangible loss in matching stats [11].
For DIA, they used the new ZT Scan DIA 2.0 approach [11,21]. ZT Scan DIA 2.0 leverages the improved sensitivity and speed of the ZenoTOF 8600 system, to deliver DIA with a narrow isolation window over a wide m/z range in less than a second, without any compromise in spectral quality, LLOQ or dynamic range of detection [3,11]. The system can adjust the Q1 isolation size from 5 to 20 Da, depending on target m/z range and desired cycle time, and perform up to 858 MS2 scans per second [11]. The ZT Scan DIA 2.0 data can be transformed to DDA-like data automatically, with apparent precursor hops of 1 to 4 Da, that is, one-fi fth of the Q1 isolation window [11]. This translates to the detection and identifi cation of 50% more lipids based on MS2 data than DDA [11]. Zamboni and his team pushed the ZT Scan DIA 2.0 even further, using a very fast, 2.2-minute LC gradient and fragmenting all the precursors in a cycle time of 400 ms, including MS1. The result was the putative annotation of 1300 lipids (see Figure 3) [3,11].
ZT Scan DIA 2.0 was also tested successfully by Zamboni to analyse the metabolomics of other complex matrices, including milk, gut extract, and serum [3]. In plants, bitter- tasting alkaloids like those in Swertia chirayita (Roxb.) may be too scarce to appear in DDA-driven MS2 scans, leaving gaps in annotation [3,11]. Thus, while DDA provides clarity for abundant molecules, it cannot overcome the problem of complexity on its own [3]. Even so, a fast DDA analysis using the ZenoTOF 8600 system on S. chirayita plant extracts produced higher quality spectra and provided 80% more annotations (see fi gure 4)—across all of the classes, compared with the ZenoTOF 7600 system [3,11]. The breakthrough though came from ZT Scan DIA 2.0 with fi ltered spectra, which proved particularly powerful, putatively identifying 50% to 100% more compounds than DDA on the ZenoTOF 8600 system (see Figure 4) [3,11].
The technological advances
The OptiFlow Pro ion source incorporates the reliability and effi ciency of the Turbo V ion source while providing fl exibility for quickly switching fl ow rates [21]. It uses an orthogonal spray and V heater design with improved geometry to enhance electrospray ionisation (ESI) droplet desolvation and sensitivity [22,23]. Its pre-optimised probe/ electrode positions and interchangeable towers allow easy switching between different ionisations (e.g., ESI and atmospheric pressure chemical ionisation [APCI]) and different fl ow rate regimes [22].
Ion transfer from the source to the mass spectrometer is critical for MS sensitivity [24]. Conventional orifi ce-and-skimmer designs scatter ions, reducing transmission, especially for low-abundance species [24]. The single-stage QJet ion guide overcomes this using ionic quadrupoles and RF fi elds: after the supersonic gas jets expand to form Mach discs, collisional and RF focusing refocus the ions into a narrow beam, enhancing
Figure 4: Analysis of metabolome extracts of the plant Swertia chirayita (Roxb.) using a fast mixed mode 3.5-minute LC reversed-phase anion-exchange method by DDA and ZT Scan DIA 2.0 (Isolation 20 Da, Q1-fi ltered). The aggregation of scores and fi nal ranking were done by tima. Three alternative scoring schemes were applied: One with stringent cutoffs, passing only high-confi dence hits supported by a strong match to the MS2 library, one with a loose scheme suited for discovery of new natural products, and one with an intermediate scheme between the two. the low-evidence scheme, With ZT Scan DIA 2.0, the results varied depending on the stringency scheme. In the low-evidence scheme, ZT Scan DIA 2.0 with Q1 fi ltering identifi ed more than four times the number of unique structures than DDA. Even without Q1 fi ltering, ZT Scan DIA 2.0 identifi ed more unique structures than DDA. These increases observed with and without Q1 fi ltering, over DDA were also observed with the intermediate- and high-stringency schemes, though to a lesser extent, which may have been curtailed due to the increasing stringency [2].
sensitivity across m/z ranges [24]. The dual-stage DJet ion guide, utilising multipoles and tapered electrodes, enhances ion focusing and transmission at higher gas pressures, thereby boosting sensitivity in systems such as the ZenoTOF 8600 system [25].
Continuous development of SWATH DIA has led to ZT Scan DIA 2.0, which leverages the Zeno trap [14, 26]. The Zeno trap boosts sensitivity by increasing duty cycle to >90% [16]. Conventional TOF systems lose the majority of ions—75–95% duty cycle—due to orthogonal injection timing mismatches [16], but the Zeno trap temporarily holds ions and releases them in synchronised bursts with the TOF pulse [16]. This improves ion detection four- to twenty-fold without sacrifi cing resolution or scan speed, capturing more MS/MS events per LC peak [16]. Speed, or with DIA equated to the number of MS/ MS per unit time, is achieved by the high-speed scanning of Q1 and TOF pulse rate. This enables over an order of magnitude more MS/MS to be acquired vs. Zeno SWATH DIA.
In ZT Scan DIA 2.0, these gains enhance metabolomics performance [16,17]. DIA fragments all ions within defi ned m/z windows, requiring high sensitivity and effi cient duty cycles to detect both abundant and low-abundance metabolites [16,17]. By maximising detected ions, the Zeno trap enables capture of scarce metabolites and
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