Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Veerasammy, Kelly; Chen, Yuki X.; Sauma, Sami; Pruvost, Mathilde; Dansu, David K; Choetso, Tenzin; Zhong, Tiffany; Maréchal, Damien; Casaccia, Patrizia; Abzalimov, Rinat R.; He, Ye

doi:10.3791/62008

Cited by 6 publications

(8 citation statements)

References 6 publications

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“…Toward this end we conducted a simple molecular screen in the fly brain using matrix-assisted laser desorption/ionization – time-of-flight (MALDI- TOF) mass spectrometry. This method supports the mass spectrometric analysis of fly head sections ( 62 ) and consists of three major steps: the crystallization of a matrix and analytes within the tissue section, the laser ionization of analytes within this matrix, and time-of-flight mass spectrometry (MS) to analyze the molecules present ( 63 ). This method provides sufficient spatial resolution to detect the presence of molecules defined by their weight over charge ratios (m/z), specifically within the central brain (Figure 6B).…”

Section: Resultsmentioning

confidence: 99%

“…After 24-h of sleep deprivation using 220s immobility triggers for focal flies with their yoked and unperturbed controls, heads from all three conditions were embedded in Optimal Cutting Temperature (Tissue-Tek, Product code: 4583, Sakura Finetek USA Inc.) compound, frozen with liquid nitrogen, cryo-sectioned, and prepared and assayed by MALDI-TOF as previously described ( 63 ). 2,5-Dihydrobenzoc acid (Sigma-Aldrich, Cat #149357) was used as our matrix, which supports the ionization of metabolites and peptides, and is considered a useful matrix for “universal analysis” of a diverse variety of molecular types ( 64 ) MALDI mass spectra were acquired in a Bruker Autoflex Speed TOF Machine (Bruker, Germany) and peaks were identified in SCiLS MALDI Imaging software (Bruker Daltonics, Germany) by a manual peak walk across the spectrum of m/z values between 0 and 1300 for each discernible m/z ratio for unperturbed, focal, and yoked flies.…”

Section: Methodsmentioning

confidence: 99%

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Homeostatic Control of Deep Sleep inDrosophila: Implications for Discovering Correlates of Sleep Pressure

Chowdhury

Abhilash

Ortega

et al. 2022

Preprint

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A defining feature of sleep is its homeostatic control, which is most clearly expressed as increased sleep after forced wakefulness. Drosophila has served as a powerful model system for understanding the homeostatic control of sleep and ongoing work continues to be an important complement to studies in mammals and other vertebrate models. Nevertheless, there are significant challenges confronting investigators of sleep regulation in Drosophila. For example, the magnitude of sleep rebound in flies is relatively modest, providing a small dynamic range over which to detect changes in homeostatic responses in experimental subjects. In addition, the perturbation necessary to keep flies awake is associated with physiological and behavioral responses that may obscure homeostatic sleep responses. Furthermore, the analysis of fly sleep as a unitary state, without differentiation between shallow and deep sleep states, clouds our ability to fully characterize homeostatic sleep responses. To address these challenges, we describe the development of a yoked-controlled paradigm for flies that allows us to produce two sets of flies that have experienced identical levels of mechanical perturbation while suffering significantly different amounts of sleep deprivation. Moreover, by differentiating long bouts of sleep from all sleep, we show that flies display significant and lasting homeostatic increases in such long bouts following sleep deprivation, that are only detectable when controlling for the sleep-independent effects of mechanical deprivation. Finally, we illustrate the importance of yoked controls for examining the molecular correlates of sleep pressure. Our work introduces methodological approaches that are likely to support the discovery of new mechanisms of sleep regulation in the fly and calls for the reevaluation of previous work identifying the molecular, physiological, and cellular correlates of sleep pressure.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Homeostatic Control of Deep Sleep inDrosophila: Implications for Discovering Correlates of Sleep Pressure

Chowdhury

Abhilash

Ortega

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Given our behavioral results indicating significant sleep-independent effects of mechanical deprivation that appear to mask homeostatic sleep responses, we sought to examine the potential utility of yoking for differentiating sleep-pressure-driven changes from the sleep-independent effects of mechanical perturbation. Toward this end we conducted a simple molecular screen in the fly brain using matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) ( Veerasammy et al, 2020 ). This method supports the MS analysis of fly head sections ( Figure 6A ; Salisbury et al, 2013 ) and consists of three major steps: the crystallization of a matrix and analytes within the tissue section, the laser ionization of analytes within this matrix, and TOF MS to analyze the molecules present ( Veerasammy et al, 2020 ).…”

Section: Resultsmentioning

confidence: 99%

Homeostatic control of deep sleep and molecular correlates of sleep pressure in Drosophila

Chowdhury,

Abhilash,

Ortega

et al. 2023

eLife

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Homeostatic control of sleep is typically addressed through mechanical stimulation-induced forced wakefulness and the measurement of subsequent increases in sleep. A major confound attends this approach: biological responses to deprivation may reflect a direct response to the mechanical insult rather than to the loss of sleep. Similar confounds accompany all forms of sleep deprivation and represent a major challenge to the field. Here, we describe a new paradigm for sleep deprivation in Drosophila that fully accounts for sleep-independent effects. Our results reveal that deep sleep states are the primary target of homeostatic control and establish the presence of multi-cycle sleep rebound following deprivation. Furthermore, we establish that specific deprivation of deep sleep states results in state-specific homeostatic rebound. Finally, by accounting for the molecular effects of mechanical stimulation during deprivation experiments, we show that serotonin levels track sleep pressure in the fly’s central brain. Our results illustrate the critical need to control for sleep-independent effects of deprivation when examining the molecular correlates of sleep pressure and call for a critical reassessment of work that has not accounted for such non-specific effects.

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“…MALDI-FTICR imaging MS is an emerging method for identifying label-free compounds and determining their spatial localization. The chemical matrix application (Nambiar et al 2021 ) and sample preparation steps (Veerasammy et al 2020 ) have a significant impact on ionization and the lateral resolution of imaging applications. Given poor matrix deposition caused by a layer of excess liquid lipids on the tissue surface, we aimed to identify a solution to achieve a uniform matrix distribution by reducing excess liquid lipids with filter paper, thereby improving the detection performance of MALDI-FTICR imaging MS in lipid-rich WAT.…”

Section: Resultsmentioning

confidence: 99%

A simple preparation step to remove excess liquid lipids in white adipose tissue enabling improved detection of metabolites via MALDI-FTICR imaging MS

Wang

Sun

Kunzke

et al. 2022

Histochem Cell Biol

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Matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) imaging mass spectrometry (MS) is a powerful technology used to analyze metabolites in various tissues. However, it faces significant challenges in studying adipose tissues. Poor matrix distribution and crystallization caused by excess liquid lipids on the surface of tissue sections hamper m/z species detection, an adverse effect that particularly presents in lipid-rich white adipose tissue (WAT). In this study, we integrated a simple and low-cost preparation step into the existing MALDI-FTICR imaging MS pipeline. The new method—referred to as filter paper application—is characterized by an easy sample handling and high reproducibility. The aforementioned filter paper is placed onto the tissue prior to matrix application in order to remove the layer of excess liquid lipids. Consequently, MALDI-FTICR imaging MS detection was significantly improved, resulting in a higher number of detected m/z species and higher ion intensities. After analyzing various durations of filter paper application, 30 s was found to be optimal, resulting in the detection of more than 3700 m/z species. Apart from the most common lipids found in WAT, other molecules involved in various metabolic pathways were detected, including nucleotides, carbohydrates, and amino acids. Our study is the first to propose a solution to a specific limitation of MALDI-FTICR imaging MS in investigating lipid-rich WAT. The filter paper approach can be performed quickly and is particularly effective for achieving uniform matrix distribution on fresh frozen WAT while maintaining tissue integrity. It thus helps to gain insight into the metabolism in WAT.

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Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Cited by 6 publications

References 6 publications

Homeostatic Control of Deep Sleep inDrosophila: Implications for Discovering Correlates of Sleep Pressure

Homeostatic Control of Deep Sleep inDrosophila: Implications for Discovering Correlates of Sleep Pressure

Homeostatic control of deep sleep and molecular correlates of sleep pressure in Drosophila

A simple preparation step to remove excess liquid lipids in white adipose tissue enabling improved detection of metabolites via MALDI-FTICR imaging MS

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