Determination of online quenching efficiency for an automated cellular microfluidic metabolomic platform using mass spectrometry based ATP degradation analysis

Filla, Laura A.; Sanders, Katherine L.; Coulton, John B; Filla, Robert T.; Edwards, John A.

doi:10.1007/s00216-019-02018-3

Cited by 8 publications

(6 citation statements)

References 26 publications

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“…Control plates were swapped to DMEM with 2% FBS. All plates were lysed after 16 h as previously described . Briefly, the medium was removed, and the cells were rapidly rinsed with 1 mL of phosphate-buffered saline.…”

Section: Methodsmentioning

confidence: 99%

Dual Fragmentation Isobaric Tags for Metabolomics

Armbruster

Grady

Caldwell

et al. 2023

J. Am. Soc. Mass Spectrom.

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Isobaric tags typically leverage an a1 type fragmentation to produce constant mass reporter ions. While this motif allows for efficient reporter formation, isobaric tags lack structural diversity, which limits the number and type of isotopes that are synthetically available. Presented here are two examples of dual fragmentation isobaric tagging. The first example mimics the typical isobaric tag structure through trimethylamine neutral loss and cyclization. Subsequent fragmentation releases a constant mass reporter with high efficiency. This provides a route to create a variety of isobaric tags with regard to both the reporter and the balancer mass. The second example is a set of six-plex isobaric, thiol-reactive tags that produce constant mass reporters by a similar sequential fragmentation mechanism. A trimethylamine neutral loss allows for the incorporation of up to 13 total isotopes in the balancer region, while minimizing deuterium retention time shifts. A subsequent C−S bond cleavage produces a constant mass reporter in the low-mass region. The thiols investigated produced an average RSD of 14% and R 2 of 0.98 when analyzed as a six-plex injection. Thiol metabolism was disrupted using the glutamyl-cysteine synthetase inhibitor buthionine sulfoximine (BSO). Endothelial cells were incubated with BSO and showed significant decreases in glutathione and cysteinyl-glycine compared to control. Overall, a new method to generate constant mass reporters using a dual fragmentation scheme is presented.

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Section: Methodsmentioning

confidence: 99%

Dual Fragmentation Isobaric Tags for Metabolomics

Armbruster

Grady

Caldwell

et al. 2023

J. Am. Soc. Mass Spectrom.

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“…An important aspect to consider in cell metabolomics is the potential degradation of the metabolites being quantified when cells are exposed to lysis processes. Filla et al [86] studied ATP degradation and developed a strategy to quench metabolic degradation illustrating the immediate need for proper quenching in metabolomic assessments.…”

Section: Metabolomics On Cellsmentioning

confidence: 99%

“…Filla et al. [86] studied ATP degradation and developed a strategy to quench metabolic degradation illustrating the immediate need for proper quenching in metabolomic assessments.…”

Section: Microfluidics For Metabolomic and Proteomic Analysis Of Cellsmentioning

confidence: 99%

Microfluidic systems in clinical diagnosis

Lapizco‐Encinas

Zhang

2022

Electrophoresis

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The use of microfluidic devices is highly attractive in the field of biomedical and clinical assessments, as their portability and fast response time have become crucial in providing opportune therapeutic treatments to patients. The applications of microfluidics in clinical diagnosis and point‐of‐care devices are continuously growing. The present review article discusses three main fields where miniaturized devices are successfully employed in clinical applications. The quantification of ions, sugars, and small metabolites is examined considering the analysis of bodily fluids samples and the quantification of this type of analytes employing real‐time wearable devices. The discussion covers the level of maturity that the devices have reached as well as cost‐effectiveness. The analysis of proteins with clinical relevance is presented and organized by the function of the proteins. The last section covers devices that can perform single‐cell metabolomic and proteomic assessments. Each section discusses several strategically selected recent reports on microfluidic devices successfully employed for clinical assessments, to provide the reader with a wide overview of the plethora of novel systems and microdevices developed in the last 5 years. In each section, the novel aspects and main contributions of each reviewed report are highlighted. Finally, the conclusions and future outlook section present a summary and speculate on the future direction of the field of miniaturized devices for clinical applications.

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“…Filla et al described a continuous-flow microfluidic device capable of automated cell lysis, metabolite extraction, and quenching of enzymatic activity. 114 Quenching efficiency was measured with an off-line MALDI-MS assay of exogenous isotopic adenosine triphosphate (ATP) hydrolysis to isotopic adenosine diphosphate (ADP), which was used as a marker of metabolite degradation. The samples processed on the chip were manually transferred onto a MALDI plate for mass spectrometry analysis.…”

Section: Coupling Of Microfluidics With Mass Spectrometrymentioning

confidence: 99%