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Simultaneous Quantitation and Discovery ( SQUAD) analysis seamlessly combines discovery and targeted workflows into a single experiment, offering a more comprehensive and versatile solution. Leveraging Thermo Scientific mass spectrometers, SQUAD workflows are enhanced by the unique hardware and software solutions of these platforms.
To enhance depth of discovery analysis, the Thermo Scientific AcquireX workflow optimizes the interrogation of relevant compounds by MS2, leading to greater coverage and more confident annotation when compared to traditional data-dependent analysis.
For robust and precise quantitative measurements with varying sensitivity to meet all application needs, Thermo Scientific mass spectrometers contain Orbitrap, Ion-Trap, and Astral detectors. Additionally, these detectors can operate in parallel configurations enabling researchers to collect all necessary metabolomics data efficiently.
For comprehensive quantitation and untargeted discovery data in a single injection, SQUAD analysis is conducted on Thermo Scientific Orbitrap Exploris Mass Spectrometers, which combine quadrupole and Orbitrap technologies. Full scan MS1 profiling with rapid polarity switching, at 60K and 120K resolution, provides sufficient points across the peak, complementing high-throughput methods.
Additionally, the AcquireX workflow for intelligent data acquisition is applied to study pool samples and maximizes the depth of metabolite identification that can be achieved in comparison to traditional data dependent MS2.
In the SQUAD workflow, sample preparation includes calibration curves and internal standards where appropriate and available for the targeted analysis portion.
Full scan MS1 profiling with polarity switching is used on all samples and AcquireX iterative data-dependent analysis is performed on the study pools. Data is then processed in two distinct ways:
For fully parallelized acquisition of both targeted and untargeted data, Orbitrap Tribrid Mass Spectrometers combine quadrupole, Orbitrap and ion trap technologies.
During SQUAD analysis on Orbitrap Tribrid Mass Spectrometers, full scan MS1 profiling is performed in the Orbitrap for untargeted discovery data acquisition while known metabolites of interest are detected using targeted-MS2 experiments with sensitive ion-trap detection.
Compared to full scan Orbitrap quantitation, targeted MS2 ion-trap methods often exhibit an additional order of magnitude linearity at the low end of the curve. At a concentration of 5 femtomoles on the column, sufficient scans of the spiked isotope-labeled phenylalanine in NIST SRM 1950 plasma were observed, achieving a linear dynamic range of six orders of magnitude.
Linear ion trap mass analyzer for accurate and sensitive targeted analysis. A sufficient number of scans of the spiked isotope-labeled phenylalanine in NIST SRM 1950 plasma were observed with 5 femto mole on the column.
Absolute quantitation results for isotope-labeled phenylalanine spike in NIST SRM 1950 plasma reference standard.
Additionally, the AcquireX workflow for intelligent data acquisition is applied to study pool samples, maximizing the range of relevant compounds interrogated by MS2. This approach increases compound identifications and reduces redundant fragmentations commonly associated with traditional data-dependent methods.
"Quantification is the next frontier in metabolomics and lipidomics. Optimal use of targeted ion-trap MS/MS time in the SQUAD approach is key for getting reliable peak intensities for low abundant compounds without compromising the untargeted full scan orbitrap discovery analysis. This advance is critically needed in the field."
Oliver Fiehn
Department of Molecular and Cellular Biology, University of California Davis
“The ability of conducting hypothesis-generating (non-targeted) and hypothesis-testing (targeted) analyses in the same LC-MS experiment is a game changer, saving us precious time and resources. Quantitative targeted ion trap data is highly robust across batches, minimizing any experimental drifts or artifacts.”
Facundo M. Fernández, PhD.
Regents’ Professor and Vasser-Woolley Chair in Bioanalytical Chemistry, School of Chemistry and Biochemistry & Petit Institute for Bioengineering & Bioscience (IBB), Georgia Institute of Technology
The Orbitrap Astral Mass Spectrometer combines quadrupole, Orbitrap, and the innovative Astral mass analyzer for high-throughput SQUAD capabilities with exceptional speed, sensitivity, and annotations. During SQUAD analysis, the Orbitrap performs full scan MS1 profiling, while the Astral detector achieves comprehensive metabolome MS2 coverage.
A single metabolomics injection on the Orbitrap Astral MS provides over 90% MS2 coverage of all compounds in the sample. This rapid performance eliminates the need to investigate only pool samples for identification, enabling in-depth MS2 analysis of all samples in your study.
On an Orbitrap Astral MS, full scan detection is done in the orbitrap while either data-dependent or targeted MS2 is performed in the Astral mass analyzer. This allows for both discovery and targeted to be collected simultaneously while exploiting the speed and sensitivity of the HRAM Astral.
A 15-minute gradient can be reduced to increase throughput by exploiting the speed of the Orbitrap Astral MS.
The number of Orbitrap MS1 scans across the plasma phenylalanine peak of 15- and 5-min LC gradients remain high, while the percentage of compound fragmentation achieved in data-dependent MS2 analysis (DDA) remains above 90%.
“The capability to generate MS/MS data for nearly all compounds in a single, short run on an Orbitrap Astral MS is a huge step forward in metabolomics. It will be particularly important for characterizing exposure chemicals, such as drugs and certain food components.”
Gary Patti, Ph.D.
Departments of Chemistry, Genetics, Medicine, Washington University in St. Louis
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