Evaluation of Data-Dependent MS/MS Acquisition Parameters for Non-Targeted Metabolomics and Molecular Networking of Environmental Samples: Focus on the Q Exactive Platform

Stincone, Paolo; Shah, Abzer K. Pakkir; Schmid, Robin; Graves, Lana G; Lambidis, Stilianos P.; Torres, Ralph R.; Xia, Shu-Ning; Minda, Vidit; Aron, Allegra T.; Wang, Mingxun; Hughes, Chambers C.; Petras, Daniel

doi:10.1021/acs.analchem.3c01202

Cited by 19 publications

(7 citation statements)

References 58 publications

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“…Furthermore, mass spectrometer settings greatly affect ion acquisition. Mass spectrometer setting optimization has been explored but is not a topic of this investigation 10 , 11 .…”

Section: Resultsmentioning

confidence: 99%

An assessment of AcquireX and Compound Discoverer software 3.3 for non-targeted metabolomics

Cooper,

Yang

2024

Sci Rep

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We used the Exploris 240 mass spectrometer for non-targeted metabolomics on Saccharomyces cerevisiae strain BY4741 and tested AcquireX software for increasing the number of detectable compounds and Compound Discoverer 3.3 software for identifying compounds by MS2 spectral library matching. AcquireX increased the number of potentially identifiable compounds by 50% through six iterations of MS2 acquisition. On the basis of high-scoring MS2 matches made by Compound Discoverer, there were 483 compounds putatively identified from nearly 8000 candidate spectra. Comparisons to 20 amino acid standards, however, revealed instances whereby compound matches could be incorrect despite strong scores. Situations included the candidate with the top score not being the correct compound, matching the same compound at two different chromatographic peaks, assigning the highest score to a library compound much heavier than the mass for the parent ion, and grouping MS2 isomers to a single parent ion. Because the software does not calculate false positive and false discovery rates at these multiple levels where such errors can propagate, we conclude that manual examination of findings will be required post software analysis. These results will interest scientists who may use this platform for metabolomics research in diverse disciplines including medical science, environmental science, and agriculture.

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“…Furthermore, mass spectrometer settings greatly affect ion acquisition. Mass spectrometer setting optimization has been explored but is not a topic of this investigation 10 , 11 .…”

Section: Resultsmentioning

confidence: 99%

An assessment of AcquireX and Compound Discoverer software 3.3 for non-targeted metabolomics

Cooper,

Yang

2024

Sci Rep

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“…It is noted that the strategy of DIA can always get the best fragmentation at (or near) the apex of the compound peak, while DDA can hardly be promised to trigger at the apex, even with the system optimization or apex-trigger algorithm. 33 This means the GlycanDIA can generally produce glycan fragmentation with good quality and higher sensitivity. For example, in our analysis of glycans on HEK 293T RNA, we identified nearly 100 glycans using only 5 μg of small RNAs, while 25 μg of HEK 293T small RNAs were used in the previous study, finding only 14 N -glycans.…”

Section: Discussionmentioning

confidence: 99%

Development and application of GlycanDIA workflow for glycomic analysis

Xie,

Liu,

Zhao

et al. 2024

Preprint

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Glycans modify protein, lipid, and even RNA molecules to form the regulatory outer coat on cells called the glycocalyx. The changes in glycosylation have been linked to the initiation and progression of many diseases. Thus, while the significance of glycosylation is well established, a lack of accessible methods to characterize glycans has hindered the ability to understand their biological functions. Mass spectrometry (MS)-based methods have generally been at the core of most glycan profiling efforts; however, modern data-independent acquisition (DIA), which could increase sensitivity and simplify workflows, has not been benchmarked for analyzing glycans. Herein, we developed a DIA-based glycomic workflow, termed GlycanDIA, to identify and quantify glycans with high sensitivity and accuracy. The GlycanDIA workflow combined higher energy collisional dissociation (HCD)-MS/MS and staggered windows for glycomic analysis, which facilitates the sensitivity in identification and the accuracy in quantification compared to conventional data-dependent acquisition (DDA)-based glycomics. To facilitate its use, we also developed a generic search engine, GlycanDIA Finder, incorporating an iterative decoy searching for confident glycan identification and quantification from DIA data. The results showed that GlycanDIA can distinguish glycan composition and isomers from N-glycans, O-glycans, and human milk oligosaccharides (HMOs), while it also reveals information on low-abundant modified glycans. With the improved sensitivity, we performed experiments to profile N-glycans from RNA samples, which have been underrepresented due to their low abundance. Using this integrative workflow to unravel the N-glycan profile in cellular and tissue glycoRNA samples, we found that RNA-glycans have specific forms as compared to protein-glycans and are also tissue-specific differences, suggesting distinct functions in biological processes. Overall, GlycanDIA can provide comprehensive information for glycan identification and quantification, enabling researchers to obtain in-depth and refined details on the biological roles of glycosylation.

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“…As reversed phase HPLC column, a Phenomenex Kinetex 1.7 µm EVO C18 RP, 100 Å pore size, dimension: 50 x 2.0 (mm, length x inner diameter) was employed. The MS/MS method was previously optimized 37 , the HESI source parameters were set as follows: auxiliary gas flow and temperature were respectively 12 AU and 400 °C, sweep gas flow was 1 L/min, sheath gas flow rate was set to 50 AU. The MS was operated in positive mode, The spray voltage was set to 3.5 kV while an inlet capillary temperature of 250 °C was adopted.…”

Section: Methodsmentioning

confidence: 99%

ModiFinder: Tandem Mass Spectral Alignment Enables Structural Modification Site Localization

Shahneh,

Strobel,

Vitale

et al. 2024

Preprint

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Untargeted tandem mass spectrometry (MS/MS) has become a high-throughput method to measure small molecules in complex samples. One key goal is the transformation of these MS/MS spectra into chemical structures. Computational techniques such as MS/MS library search have enabled the re-identification of known compounds. Analog library search and molecular networking extend this identification to unknown compounds. While there have been advancements in metrics for the similarity of MS/MS spectra of structurally similar compounds, there is still a lack of automated methods to provide site specific information about structural modifications. Here we introduce ModiFinder that leverages the alignment of peaks in MS/MS spectra between structurally related known and unknown small molecules. Specifically, ModiFinder focuses on shifted MS/MS fragment peaks in the MS/MS alignment. These shifted peaks putatively represent substructures of the known molecule that contain the site of the modification. ModiFinder synthesizes these information together and scores the likelihood for each atom in the known molecule to be the modification site. We demonstrate in this manuscript how ModiFinder can effectively localize modifications which extends the capabilities of MS/MS analog searching and molecular networking to accelerate the discovery of novel compounds.

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Evaluation of Data-Dependent MS/MS Acquisition Parameters for Non-Targeted Metabolomics and Molecular Networking of Environmental Samples: Focus on the Q Exactive Platform

Cited by 19 publications

References 58 publications

An assessment of AcquireX and Compound Discoverer software 3.3 for non-targeted metabolomics

An assessment of AcquireX and Compound Discoverer software 3.3 for non-targeted metabolomics

Development and application of GlycanDIA workflow for glycomic analysis

ModiFinder: Tandem Mass Spectral Alignment Enables Structural Modification Site Localization

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