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Enhanced quantification of metabolic activity for individual adipocytes by label-free FLIM.

ABSTRACT: Fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores such as nicotinamide adenine dinucleotide (NADH) allows for label-free quantification of metabolic activity of individual cells over time and in response to various stimuli, which is not feasible using traditional methods due to their destructive nature and lack of spatial information. This study uses FLIM to measure pharmacologically induced metabolic changes that occur during the browning of white fat. Adipocyte browning increases energy expenditure, making it a desirable prospect for treating obesity and related disorders. Expanding from the traditional two-lifetime model of NADH to a four-lifetime model using exponential fitting and phasor analysis of the fluorescence decay results in superior metabolic assessment compared to traditional FLIM analysis. The four lifetime components can also be mapped to specific cellular compartments to create a novel optical ratio that quantitatively reflects changes in mitochondrial and cytosolic NADH concentrations and binding states. This widely applicable approach constitutes a powerful tool for studies where monitoring cellular metabolism is of key interest.

SUBMITTER: Evers M

PROVIDER: S-EPMC5993796 | biostudies-other | 2018 Jun

REPOSITORIES: biostudies-other

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Enhanced quantification of metabolic activity for individual adipocytes by label-free FLIM.

Evers Michael M Salma Nunciada N Osseiran Sam S Casper Malte M Birngruber Reginald R Evans Conor L CL Manstein Dieter D

Scientific reports 20180608 1

Fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores such as nicotinamide adenine dinucleotide (NADH) allows for label-free quantification of metabolic activity of individual cells over time and in response to various stimuli, which is not feasible using traditional methods due to their destructive nature and lack of spatial information. This study uses FLIM to measure pharmacologically induced metabolic changes that occur during the browning of white fat. Adipocyte browning ...[more]

PMID: 29884881

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Project description:Despite data-independent acquisition (DIA) has been increasingly used for relative protein quantification, DIA-based label-free absolute quantification method has not been fully established. Here we present a novel DIA method using the TPA algorithm (DIA-TPA) for the absolute quantification of protein expressions in human liver microsomal and S9 samples. To validate this method, both data-dependent acquisition (DDA) and DIA experiments were conducted on 36 individual human liver microsome and S9 samples. The MS2-based DIA-TPA was able to quantify approximately twice as many proteins as the MS1-based DDA-TPA method, whereas protein concentrations determined by the two approaches were comparable. To evaluate the accuracy of the DIA-TPA method, we absolutely quantified carboxylesterase 1 concentrations in human liver S9 fractions using an established SILAC internal standard-based proteomic assay; the SILAC results were consistent with those obtained from DIA-TPA analysis. Finally, we employed a unique algorithm in DIA-TPA to distribute the MS signals from shared peptides to individual proteins or isoforms and successfully applied the method to the absolute quantification of several drug-metabolizing enzymes in human liver microsomes. In sum, the DIA-TPA method not only can absolutely quantify entire proteomes and specific proteins, but also has the capability quantifying proteins with shared peptides. SIGNIFICANCE: Data independent acquisition (DIA) has emerged as a powerful approach for relative protein quantification at the whole proteome level. However, DIA-based label-free absolute protein quantification (APQ) method has not been fully established. In the present study, we present a novel DIA-based label-free APQ approach, named DIA-TPA, with the capability absolutely quantifying proteins with shared peptides. The method was validated by comparing the quantification results of DIA-TPA with that obtained from stable isotope-labeled internal standard-based proteomic assays.

Dataset Information

Enhanced quantification of metabolic activity for individual adipocytes by label-free FLIM.

Publications

Enhanced quantification of metabolic activity for individual adipocytes by label-free FLIM.

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