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

• Published in: Analytical and bioanalytical chemistry Vol. 411; no. 24; pp. 6399 - 6407
• Main Authors: Filla, Laura A., Sanders, Katherine L., Coulton, John B., Filla, Robert T., Edwards, James L.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.09.2019; Springer; Springer Nature B.V
• Subjects: Adenosine diphosphate; Adenosine Diphosphate - metabolism; Adenosine triphosphatase; Adenosine triphosphate; Adenosine Triphosphate - metabolism; Analytical Chemistry; ATP; Automation; Biochemistry; Biotechnology; Cell culture; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; cold treatment; Degradation; Enzymatic activity; enzyme activity; Flow rates; Flow velocity; Food Science; Histidine; Hydrolysis; Intracellular; Laboratory Medicine; liquid chromatography; Lysis; Mass spectrometry; Mass spectroscopy; metabolism; Metabolites; Metabolomics; Microfluidic devices; Microfluidics; Microfluidics - methods; Monitoring/Environmental Analysis; Muscle proteins; N-Acetylglucosamine; Optimization; Perturbation; Preservation; Pyruvic acid; Quenching; Reliability analysis; Research Paper; Sample preparation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Technology application; temperature; Temperature control; Tryptophan; Massachusetts; Metabolomics; Sample preparation; Mass spectrometry; Adenosine triphosphate; Microfluidic; Quenching
• ISSN: 1618-2642; 1618-2650; 1618-2650
• DOI: 10.1007/s00216-019-02018-3

As microfluidic cell culture progresses, the need for robust and reproducible intracellular analyses grows. In particular, intracellular metabolites are subject to perturbation and degradation during the lysing process. The reliability of intracellular metabolomic analysis in microfluidic devices depends on the preservation of metabolite integrity during sample preparation and storage. Described here is a novel automated microfluidic system exhibiting the necessary rapid cellular lysis and quenching of enzymatic activity. Quenching efficiency was assessed using a novel ratiometric MALDI-MS-based assay of exogenous isotopic adenosine triphosphate (ATP) hydrolysis to isotopic adenosine diphosphate (ADP) as a marker of metabolite degradation. The lysis system of the microfluidic device was enhanced using a Peltier cooler to chill the lysate and quench aberrant enzymatic activity. Parameter optimization (flow rate, collection time, and temperature control) improved the endogenous and exogenous ADP/ATP ratios by 44.9% and 39.8% respectively consistent with traditional quenching techniques. The effects of chilling/quenching on metabolism were evaluated resulting in over 500 significant features compared to non-chilled from untargeted capillary LC-MS metabolomic analyses. These include increased levels of tryptophan, histidine, and pyruvate as well as decreased levels in UDP- N -acetylglucosamine. The results illustrate the need for both rapid lysis and quenching in microfluidic cell culture platforms. Graphical abstract