Accelerate Discovery with SWATH DIA Mass Spectrometry

Accelerating Discovery Using SWATH DIA Mass Spectrometry 1 Mass spectrometry (MS) enables the precise detection and quantitation of compounds, often involving fragmentation of ions to infer details about parent molecules with great specificity. Thanks to continuous innovations, MS has become one of the most valuable analytical tools and has multiple applications in life science research. In this article, Katherine Tran, senior manager for global strategic marketing in proteomics science research at SCIEX, presents the latest MS technology and discusses how it can be used to enhance research. Data acquisition methods Shotgun, or bottom-up, MS is a key method used in life science studies. It allows researchers to make a global interpretation of a biological system by comprehensively analyzing all its compounds and its environment. There are two main approaches to generate tandem mass spectra: data dependent acquisition (DDA) – which is also known as information dependent acquisition (IDA) – and SWATH data independent acquisition (DIA). DDA is a well-established method used for many applications; it provides untargeted sample analysis by selecting and fragmenting multiple charge ions from a full scan mass spectrum to generate tandem MS/MS mass spectra. While DDA ensures selectivity, it creates problems in large-scale sample series and fast proteomic measurements as the analysis only measures a select number of the ion parents present and consequently means values for some species will be missing. A small shift in retention time from run to run can affect the population of precursor ions entering the instrument during each cycle therefore, the subset of compounds that are analyzed, leading to inter-experimental variations. Although the ability to ionize and detect has significantly improved, the matrices remain just as complex, and the instrument does not have time to sample all the potential precursors entering the system. To counter this, a target list of precursors can be used; however, this negates the benefit of a truly unbiased and global approach. Additionally, when comparing multiple DDA datasets, missing peaks and gaps are often observed, which can be particularly impactful for lower-level analytes and low replicate numbers. The data independent strategy (SWATH DIA) – first published in 2012 – analyzes all ionizable precursors in a specified mass range by MS/MS regardless of the abundance or other criteria. This method generates a complete data set that Accelerating Discovery Using SWATH DIA Mass Spectrometry Accelerating Discovery Using SWATH DIA Mass Spectrometry 2 Accelerating Discovery Using SWATH DIA Mass Spectrometry contains fragment data for all precursor ions, resulting in greater reproducibility. Additionally, wider precursor selection windows are used, allowing simultaneous transmission of multiple precursors stepped across the entire precursor mass range, ensuring that all precursor masses are fragmented for every cycle. MS/MS spectra generated by SWATH DIA are typically more complex compared with those generated by DDA. To increase specificity, SWATH DIA can be performed with smaller, variable-sized windows to minimize the number of precursors within each window. Fast MS/MS scanning then allows iteration through all windows within each cycle while still covering a broad mass range. The use of variable window widths further increases selectivity of SWATH DIA, as the width of each precursor is adjusted according to the data complexity scanned within that mass range. Very narrow windows are used where analyte density is the greatest and wider windows are used where analytes are more sparsely populated. This increases the percentage of high-quality precursors that are identified and quantified. The Zeno revolution High sensitivity is also an important feature for mass spectrometers used in life science research. The ZenoTOF 7600 system is the latest high-resolution accurate MS system, offering significant sensitivity gains, giving richer, more comprehensive and reliable MS/MS data sets. The ZenoTOF 7600 system is a quadrupole time-of-flight mass spectrometer (Q-TOF) with the following unique features (Figure 1): 1. A “Zeno trap”, which increases duty cycle and consequently MS/MS sensitivity, allowing users to gain more information from every experiment – even when dealing with low abundance species. This can be coupled with either collision-induced dissociation (CID) or electron activated dissociation (EAD) fragmentations. 2. A new Q0 design that improves ion transmission and maintenance. 3. An EAD cell that releases electrons which fragment analytes over a desired range of energies before they are sent to the detector. The EAD cell is highly tunable, offering alternative loads of fragmentation, increased sensitivity and coverage of data for all kinds of molecules. 4. A fast and sensitive detector with a wide dynamic range. This new detector offers speeds of up to 133 Hz with five orders of linear dynamic range without compromising speed or resolution. It is also cartridge based; therefore, it can be easily replaced if needed. Increasing the duty cycle using the Zeno trap Duty cycle refers to the percentage of ions that are pulsed into the TOF region. As ions are pushed into the TOF accelerator, a proportion of ions are lost and only 5–25% of the total ions hit the TOF detector with most quadrupoleTOF systems (Figure 2 A). In contrast, the ZenoTOF 7600 system controls the ion beam as it travels from the collision cell into the TOF accelerator. Ions exit the Zeno trap in an ordered release based on potential energy (from higher Figure 1: The ZenoTOF 7600 system. Accelerating Discovery Using SWATH DIA Mass Spectrometry 3 Accelerating Discovery Using SWATH DIA Mass Spectrometry m/z species to lower m/z species), so that they all arrive at the TOF accelerator at the same time and location. The ion pocket is then pushed through the top accelerator, increasing the duty cycle to more than 95% (Figure 2 B). Advantages of the Zeno SWATH DIA workflow The Zeno SWATH DIA workflow combines the reproducibility and precision of the SWATH DIA acquisition method with the gains in sensitivity offered by the Zeno trap to deliver maximal information from each sample. Figure 3 shows that combining nanoflow chromatography data with Zeno SWATH DIA significantly improves the signal intensity compared to the standard SWATH DIA workflow. The Zeno SWATH DIA workflow can also be used to perform fast and sensitive large-scale proteomic studies using higher flow rates and at low sample loading. For example, using A. Typical Q-TOF instrument B. ZenoTOF 7600 system Figure 2. Comparison of duty cycle in a typical Q-TOF instrument (A) and the ZenoTOF 7600 system (B). Duty cycle 5–25% Duty cycle >95% Accelerating Discovery Using SWATH DIA Mass Spectrometry 4 Accelerating Discovery Using SWATH DIA Mass Spectrometry the regular SWATH DIA workflow on a 5 ng load of K562 cell lysate, it is possible to identify ~2,400 protein groups at a 1% false discovery rate (FDR) – almost 1,300 of those proteins are quantifiable when filtered at less than a 20% coefficient of variation (CV) (Figure 4). However, using the Zeno SWATH DIA workflow, it is possible to identify over 4,200 protein groups (over 2,700 of which are quantifiable when filtered at less than a 20% CV). This represents an increase of 75% for the total protein groups identified and a 108% increase for those that are quantifiable at 20% CV (Figure 4). It is also possible to achieve the same level of sensitivity of nanoflow experiments using the Zeno SWATH DIA workflow for high flow LC-MS/MS experiments – even though they typically require higher sample loading. For example, by running 5 µl/min with a 20-minute gradient, it is possible to identify about 1,400 protein groups. When the same experiment is repeated using a 5-minute gradient, it is possible to identify Figure 3: Summed signal of cell lysate data run with nanoflow chromatography (left panel: 25 ng, right panel, 200 ng). Dark green curves: data obtained using SWATH DIA. Light green curves: data obtained using Zeno SWATH DIA. Figure 4: Comparison of proteomic analysis of a K562 cell lysate using the SWATH DIA (5 ng load) or the Zeno SWATH DIA (5–200 ng) workflows. Accelerating Discovery Using SWATH DIA Mass Spectrometry 5 Accelerating Discovery Using SWATH DIA Mass Spectrometry around 80% of the protein groups found using the 20-minute gradient, even when data is filtered at less than a 20% CV (Figure 5). Conclusion Zeno SWATH DIA harnesses the precision of the SWATH DIA acquisition method and the sensitivity of the Zeno trap technology to provide the greatest depth of coverage and amount of information in the shortest amount of time. Watch the complete “Teach Me in 10” interview with Katherine Tran here Learn more about the ZenoTOF 7600 system Learn more about SWATH DIA Figure 5: Protein groups identified using the Zeno SWATH DIA workflow in a high flow LC-/MS/MS experiment at 20-minute or 5-minute gradients (left and right panels, respectively).