LC/ESI-MS/MS detection of FAs by charge reversal derivatization with more than four orders of magnitude improvement in sensitivity

Bollinger, James G.; Rohan, Gajendra; Sadı́lek, Martin; Gelb, Michael H.

doi:10.1194/jlr.d040782

Cited by 105 publications

(112 citation statements)

References 33 publications

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“…when no adipocytes were in the chip) and therefore not further investigated. Previous studies have also reported background signal from 16:0 and 18:0 in fresh LC solvents, suggesting they may represent a ubiquitous background contaminant [54]. …”

Section: Resultsmentioning

confidence: 99%

Monitoring cell secretions on microfluidic chips using solid-phase extraction with mass spectrometry

et al. 2016

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Microfluidics is an enabling technology for both cell biology and chemical analysis. We combine these attributes with a microfluidic device for on-line solid-phase extraction (SPE) and mass spectrometry (MS) analysis of secreted metabolites from living cells in culture on the chip. The device was constructed with polydimethylsiloxane (PDMS) and contains a reversibly-sealed chamber for perfusing cells. A multilayer design allowed a series of valves to control an on-chip 7.5 μL injection loop downstream of the cell chamber with operation similar to a 6-port valve. The valve collects sample and then diverts it to a packed SPE bed that was connected in-line to treat samples prior to MS analysis. The valve allows samples to be collected and injected onto the SPE bed while preventing exposure of cells to added back-pressure from the SPE bed and organic solvents needed to elute collected chemicals. Here, cultured murine 3T3-L1 adipocytes were loaded into the cell chamber and non-esterified fatty acids (NEFAs) that were secreted by the cells were monitored by SPE-MS at 30 min intervals. The limit of detection for a palmitoleic acid standard was 1.4 μM. Due to the multiplexed detection capabilities of MS, a variety of NEFAs were detected. Upon stimulation with isoproterenol and forskolin, secretion of select NEFAs was elevated an average of 1.5-fold compared to basal levels. Despite the 30 min delay between sample injections, this device is a step towards a miniaturized system that allows automated monitoring and identification of a variety of molecules in the extracellular environment.

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Section: Resultsmentioning

confidence: 99%

Monitoring cell secretions on microfluidic chips using solid-phase extraction with mass spectrometry

et al. 2016

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“…Using chemical ionization can overcome this and also allows locating double bonds in PUFAs [90,91]. A novel LC-MS based method might replace this approach in future, offering higher sensitivity using a charge reversal derivatization of free fatty acids with N-(4-aminomethylphenyl)pyridinium, although not applied to C. elegans yet [92]. The analysis of the fatty acid composition of intact lipids is getting more important, because there is only weak correlation between bound and free fatty acids.…”

Section: Mass Spectrometry Based Methodsmentioning

confidence: 99%

The Caenorhabditis elegans lipidome

Witting

Schmitt‐Kopplin

2016

Archives of Biochemistry and Biophysics

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“…Parallel incubations were made with lipoproteins alone. Lipidomic analyses, i.e., sample extractions for lysophospholipids and NEFAs, derivatization of NEFAs, and quantification by using liquid chromatography-tandem mass spectrometry (LC-MS/MS), were performed as described previously (31,32).…”

Section: Methodsmentioning

confidence: 99%

In Vitro Anti-Plasmodium falciparum Properties of the Full Set of Human Secreted Phospholipases A ₂

et al. 2015

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We have previously shown that secreted phospholipases A 2 (sPLA 2 s) from animal venoms inhibit the in vitro development of Plasmodium falciparum, the agent of malaria. In addition, the inflammatory-type human group IIA (hGIIA) sPLA 2 circulates at high levels in the serum of malaria patients. However, the role of the different human sPLA 2 s in host defense against P. falciparum has not been investigated. We show here that 4 out of 10 human sPLA 2 s, namely, hGX, hGIIF, hGIII, and hGV, exhibit potent in vitro anti-Plasmodium properties with half-maximal inhibitory concentrations (IC 50 s) of 2.9 ؎ 2.4, 10.7 ؎ 2.1, 16.5 ؎ 9.7, and 94.2 ؎ 41.9 nM, respectively. Other human sPLA2s, including hGIIA, are inactive. The inhibition is dependent on sPLA 2 catalytic activity and primarily due to hydrolysis of plasma lipoproteins from the parasite culture. Accordingly, purified lipoproteins that have been prehydrolyzed by hGX, hGIIF, hGIII, and hGV are more toxic to P. falciparum than native lipoproteins. However, the total enzymatic activities of human sPLA 2 s on purified lipoproteins or plasma did not reflect their inhibitory activities on P. falciparum. For instance, hGIIF is 9-fold more toxic than hGV but releases a lower quantity of nonesterified fatty acids (NEFAs). Lipidomic analyses of released NEFAs from lipoproteins demonstrate that sPLA 2 s with anti-Plasmodium properties are those that release polyunsaturated fatty acids (PUFAs), with hGIIF being the most selective enzyme. NEFAs purified from lipoproteins hydrolyzed by hGIIF were more potent at inhibiting P. falciparum than those from hGV, and PUFA-enriched liposomes hydrolyzed by sPLA 2 s were highly toxic, demonstrating the critical role of PUFAs. The selectivity of sPLA 2 s toward lowand high-density (LDL and HDL, respectively) lipoproteins and their ability to directly attack parasitized erythrocytes further explain their anti-Plasmodium activity. Together, our findings indicate that 4 human sPLA 2 s are active against P. falciparum in vitro and pave the way to future investigations on their in vivo contribution in malaria pathophysiology.

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LC/ESI-MS/MS detection of FAs by charge reversal derivatization with more than four orders of magnitude improvement in sensitivity

Cited by 105 publications

References 33 publications

Monitoring cell secretions on microfluidic chips using solid-phase extraction with mass spectrometry

Monitoring cell secretions on microfluidic chips using solid-phase extraction with mass spectrometry

The Caenorhabditis elegans lipidome

In Vitro Anti-Plasmodium falciparum Properties of the Full Set of Human Secreted Phospholipases A ₂

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LC/ESI-MS/MS detection of FAs by charge reversal derivatization with more than four orders of magnitude improvement in sensitivity

Cited by 105 publications

References 33 publications

Monitoring cell secretions on microfluidic chips using solid-phase extraction with mass spectrometry

Monitoring cell secretions on microfluidic chips using solid-phase extraction with mass spectrometry

The Caenorhabditis elegans lipidome

In Vitro Anti-Plasmodium falciparum Properties of the Full Set of Human Secreted Phospholipases A 2

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In Vitro Anti-Plasmodium falciparum Properties of the Full Set of Human Secreted Phospholipases A ₂