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Achieve comprehensive MSn data coverage
Redefine small-molecule research to maximize relevant data acquisition providing valuable knowledge to confidently answer complex questions. The Thermo Scientific AcquireX data acquisition workflow extends intelligence-driven mass spectrometry through experimental connectivity by integrating independent experiments into an automated workflow that enhances real-time, selective LC-MSn data acquisition for efficient and comprehensive sample and study characterization. With five different routines, the AcquireX data acquisition workflow extends productivity to all small-molecule applications, from comprehensive structural annotation to screening.
Learn more about the latest advances in data acquisition that allow you to capture more meaningful data faster than ever before.
Key benefits of AcquireX data acquisition
| Collect more meaningful data, not just more data | Fully automated, all-inclusive data acquisition | Increased productivity and more confident identifications | Maximize confident compound identification and structural annotation |
|---|---|---|---|
| Typical LC-MSn workflows require compromise among spectral quality, specificity, and coverage. Now you can break those dependencies and capture high-quality MSn spectral data on every compound in your sample, automatically. | Eliminate manual interrogation of your data to identify background ions for exclusion, or to create inclusion lists. Through automated creation of comprehensive background exclusion and sample compound inclusion lists, the breadth and depth of coverage for compounds of interest can be easily extended. | Multiple AcquireX workflow acquisition modes to extend experimental connectivity across the entire study by managing sample acquisition, exclusion or exclusion/inclusion list creation, and modifying data-dependent acquisition (DDA) methods with the sample-specific exclusion and inclusion list. | Compared to “traditional” data-dependent acquisition (DDA), both the number of compounds with in-depth fragmentation spectra and the confidence in spectral matches increases when using AcquireX-DDA. Interrogate your samples more deeply with more confidence in assignments through a range of powerful data processing capabilities. |
Learn more about the AcquireX data acquisition workflow
The comprehensive LC-MS analysis of complex samples for small-molecule identification and structural elucidation can be a bottleneck, involving tedious manual data interpretation as well as complex instrument set up and method refinement.
Successful small-molecule sample characterization requires comprehensive, accurate and sensitive detection of all relevant compounds, ideally accompanied with intelligence-driven data acquisition workflows and comprehensive data analysis tools. The workflow also needs to be flexible enough to adapt to the vast needs of small-molecule research markets while maintaining experimental efficiency and effectiveness.
Acquisition techniques such as data-independent acquisition (DIA) can ensure that fragments are generated for each compound, but due to potential overlap of multiple components fragments (especially in samples with complex matrices), even with the use of complex data deconvolution algorithms you can never be completely sure that a given fragment ion relates back to a given precursor. Conversely, with data-dependent acquisition (DDA), you can be sure that each fragment obtained can be related back to a specific precursor, indeed the resulting MS/MS spectrum contains only fragments from the precursor. However, ensuring coverage of relevant compounds can require tedious manual creation of exclusion and inclusion lists which takes time.
Using DDA with the Deep Scan AcquireX workflow can significantly increase the number of unique compounds with high-quality fragmentation spectra, so you obtain a more comprehensive picture of what is in your samples, as well as increase the depth of decision-making MS/MS information available.
Acquiring more data is only one part of the story though. Combining the AcquireX data acquisition workflow with other enabling tools means a dramatic reduction in the number of compounds without MS/MS spectra and a significant increase in the number of compounds with confident identification and ranked putative identifications. Using Thermo Scientific Compound Discoverer software in combination with a range of spectral libraries (such as Thermo Scientific mzCloud mass spectral fragmentation library), compound databases, and ranking tools (Thermo Scientific mzLogic algorithm) to aid unknown putative identification through a combination of using extensive spectral library fragmentation information and structural databases, provides higher confidence in assignments for more compounds in less time.
Using DDA with the AcquireX data acquisition workflow improves data quality and creates a significant increase in the number of compounds with MS/MS spectra, resulting in improved mzLogic ranking and higher mzCloud similarity scores, ultimately providing higher overall confidence in compound identification and putative unknown identification.
Featured videos
Choose from a range of pre-defined, single-click templates arranged by application and specific to each MS, or rapidly customize your own with drag-and-drop capabilities.
Regardless of how you choose to acquire your data, the method set-up is quick and simple, even with advanced workflows for profiling, identifying and characterizing your samples.
The AcquireX data acquisition workflow enhances intelligence-driven mass spectrometry to better determine in real-time which precursors to select for enhanced MS/MS or MSn (MS2, MS3, and/or MS4) interrogation (depending upon which mass spectrometer is being used), how to fragment the precursor to generate structurally diagnostic product ions, and how to connect one experiment to another for effective study evaluation.
With four data acquisition workflows, experimental flexibility to match study objectives ensures successful non-targeted small-molecule sample MSn analyses. Depending upon the specific workflow being selected (see the below table), the AcquireX data acquisition workflow can automate:
- Acquisition of full scan, high-resolution, accurate-mass (HRAM) MS data to create inclusion and/or exclusion lists
- Intelligent precursor selection for tandem MS acquisition based upon exclusion and/or inclusion lists
- For iterative workflows, the dynamic management of exclusion lists
AcquireX Data Acquisition Workflow
| Background Exclusion | Background Exclusion Component Inclusion | Iterative Precursor Exclusion | Deep Scan | Advanced Deep Scan | |
|---|---|---|---|---|---|
| Creation of study-specific exclusion list |  | | | | |
| Creation of sample-specific inclusion list | | | | ||
| Iterative sample analysis | | | | ||
| Dynamic exclusion list management | | | | ||
| Automated analysis of multiple samples in a study | | | | ||
| Utilizes instrumental method templates | | | | | |
Bypass the unwanted data acquisition of background matrix ions to preferentially drive acquisition of sample-specific precursors in one sample, or across a batch of samples, using the AcquireX background exclusion workflow. Incorporating comprehensive background exclusion increases the probability of low-level precursor selection within the chromatographic peak width. DDA methods are based on acquiring HRAM MS and tandem MS data within a user-defined cycle time; this cycle-time is based on the chromatographic peak width to ensure enough DDA cycles are acquired for accurate profiling, therefore balancing the amount of time dedicated for HRAM MS acquisition and that of the subsequent MSn spectra preferentially acquired.
Representation of LC-MS/MS analysis of a sample using the AcquireX background exclusion method. A narrow mass range from the full scan HRAM MS is shown to highlight the selectivity of the AcquireX method. Real-time data analysis identifies precursor m/z values on the exclusion list (shown in red) demonstrating the extended tandem MS coverage using a top 6 method, extending the dynamic range of precursor interrogation in one DDA cycle.
Like the AcquireX background exclusion workflow, the background exclusion and component inclusion workflow creates an exclusion list based upon the HRAM MS data acquisition of a representative blank or matrix sample, but also creates an inclusion list for each sample or a representative pooled sample.
The background extraction and component inclusion workflow leverages enhanced intelligence, enabling the selection of preferred ions (for example [M+H]+ adducts) from which to acquire tandem MS data. In real-time, the MS analysis can identify both [M+H]+ and [M+Na]+ ions and bypass adduct fragmentation regardless of measured ion intensity; this identification and grouping of precursors belonging to a specific compound further increases the intelligent selectivity and experimental efficiency, increasing the likelihood of increased sample coverage for compounds with high-quality fragmentation spectra.
As for the background exclusion workflow, the iterative precursor workflow creates an exclusion list based upon the HRAM MS analysis of a representative blank sample. AcquireX-DDA analysis of the sample is performed, with the exclusion list being automatically updated based upon the precursor ions being selected; additional iterative sample analyses can be performed with the exclusion list being continually updated and the process repeated until a user-defined number of replicate injections have been performed, or no more precursor ions exist for analysis.
The deep scan workflow manages replicate sample analysis and automated creation of exclusion and inclusion lists with dynamic modification of these inclusion and exclusion lists in between each replicate injection. The deep scan AcquireX workflow also has the capability to compare the lists in real-time and handle overlapping precursor m/z values to determine if it should be considered for tandem MS acquisition based on the relative ion intensity difference between the matrix blank and the sample; if the precursor ion intensity is greater in the sample by a user-defined parameter, acquisition of the tandem MS is performed.
The stepwise acquisition routine for the deep scan workflow performs precursor mapping of the solvent/matrix blank and sample to create the exclusion and inclusion lists. Following data acquisition of the first replicate injection, the data is processed to determine which precursors on the inclusion list were selected and move them to the exclusion list; this process is continued for either the user-defined number of replicate injections or the inclusion list has been exhausted.
Advanced Deep Scan AcquireX workflow provides the ultimate coverage for small-molecule analysis expanding the Deep Scan workflow with increased ease-of-use and the flexibility to submit several experiments from a single sequence automatically acquire and combine multiple blank or exclusion lists using improved algorithms for background exclusion/component detection. This is useful as an alternative to pooled matrix blanks which dilute low abundant compounds. Additionally, the user interface has been enhanced to increase ease-of-use with copy/fill-down, export/import sequence, and insert blank/wash functionalities
Now that you have acquired comprehensive MSn data for all your compounds, you need to identify them.
Combining the world’s largest mass spectral fragmentation library, mzCloud, with other online databases and the unknown identification capabilities of the mzLogic data analysis algorithm, means that you can be confident in assigning structures to your unknowns.
With both Compound Discoverer software and Thermo Scientific Mass Frontier software, you make the most of your data and automate the identification of your unknowns, even when there is no spectral match.
Whilst the acquisition of MS/MS data is usually sufficient for the confident structural assignment of many compounds when using mass spectral libraries, such as mzCloud, there can be certain studies or classes of compounds where additional levels of MSn fragmentation can be critical in differentiating between structural isomers for example.
In the example below, the MS/MS of two structural isomer flavonoids is identical, meaning that accurate structural identification is impossible. However, the AcquireX data acquisition workflow automatically triggers neutral loss MS3 results in two different fragmentation spectra, allowing for the correct distinction between the two isomers.
Comparative MS/MS spectra for two flavonoid structrual isomers where the only structural difference is associated with aglycone substructure. The addition of the two sugar rings to the base aglycone substructure results in similar MS/MS spectral patterns prohibiting correct structural identification despite low mass errors for precursor and MS/MS product ions. Incorporation of neutral-loss triggered MS3 performs tandem mass spectral analysis and subsequent MS3 acquisition resulting in clearly defining the two isomeric compounds.
 |  |  | |
| Thermo Scientific Orbitrap IQ-X Tribrid mass spectrometer | Thermo Scientific Orbitrap Exploris 480 mass spectrometer | Thermo Scientific Orbitrap Exploris 240 mass spectrometer | |
|---|---|---|---|
| Mass Accuracy | < 3 ppm RMS external calibration < 1 ppm RMS internal calibration | ||
| Resolution | Up to 500,000 FWHM at m/z 200 optional up to 1,000,000 FWHM at m/z 200 | Up to 480,000 FWHM at m/z 200 | Up to 240,000 FWHM at m/z 200 |
| MS/MS | Yes | ||
| MSn | Yes, n = 1 to 10 | No | |
| Fragmentation | CID and HCD optional UVPD | HCD | |
| Scan Rates | Up to 40 Hz for Orbitrap MSn Up to 45 Hz for Ion trap MSn | Up to 40 Hz for Orbitrap MS/MS | Up to 22 Hz for Orbitrap MS/MS |
| Orbitrap IQ-X Tribrid MS Specification Sheet | Orbitrap Exploris 480 MS Specification Sheet | Orbitrap Exploris 240 MS Specification Sheet | |
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Break your bottlenecks
Addressing the "Identity Crisis" in Small-Molecule Compound Identification
In this webinar we discuss a fundamental new approach to untargeted small-molecule analysis involving optimized mass spectrometers, powerful new data acquisition strategies, and an arsenal of new software tools to translate high-quality Orbitrap mass spectra into more confidently-assigned small-molecule structures.
Look inside the novel Orbitrap IQ-X Tribrid mass spectrometer.
