Comparative quantitative fatty acid analysis of triacylglycerols using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry and gas chromatography

Hlongwane, Caswell; Delves, I. G.; Wan, L. W.; Ayorinde, Folahan O.

doi:10.1002/rcm.462.abs

Cited by 24 publications

(35 citation statements)

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“…Furthermore, the data obtained point to the fact that sample preparation and data acquisition conditions need to be carefully controlled to obtain optimal results. In a more general context, our data, along with some other recent publications (Hlongwane et al, 2001;Bucknall et al, 2002;Mims and Hercules, 2003;Alterman et al, 2004;Helmke et al, 2004), suggest that application of MALDI-TOF mass spectrometry for relative or direct quantitation is a valid alternative to the stable isotope approach. Particularly advantageous in direct quantitation by MALDI TOF is the simplicity of the sample processing.…”

Section: Maldi Tof-based Quantitation Of P450ssupporting

confidence: 65%

“…At the same time, the number of examples of the direct use of MALDI-TOF MS without introduction of stable isotopes for quantitative analysis of biomolecules is limited (Walker et al, 2000;Hlongwane et al, 2001;Bucknall et al, 2002;Helmke et al, 2004). Working with minute amounts of biological sample obtained from liver, brain, kidney, or any other organ would benefit from a simpler quantitative approach.…”

Section: Resultsmentioning

confidence: 99%

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Quantitative Analysis of Cytochrome P450 Isozymes by Means of Unique Isozyme-Specific Tryptic Peptides: A Proteomic Approach

Alterman¹,

Kornilayev²,

Duzhak³

et al. 2005

Drug Metab Dispos

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ABSTRACT:A novel matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry method has been developed to quantitate cytochrome P450 (P450) isozymes based on their unique isozyme-specific tryptic peptides. It was shown that the molar ratio of P450 isozyme-specific peptides is linearly proportional to the mass peak area ratio of corresponding peptides not only in simple two-peptide mixtures, but also in complex digest mixtures. This approach is applicable both to in-gel (as shown for CYP2B1 and CYP2B2) and in-solution digests (as shown for CYP1A2, CYP2E1, and CYP2C19) and does not require introduction of stable isotopes or labeling with isotope-coded affinity tagging. The relative and absolute quantitation can be performed after developing corresponding calibration curves with synthesized P450 isozyme-specific peptide standards. The absolute quantitation of human P450 isozymes was performed by using CYP2B2 isozyme-specific peptide (1306.7 Da) as the universal internal standard. The utility of this approach was demonstrated for two highly homologous (>97%) rat liver CYP2B1 and CYP2B2 and three human P450 isozymes belonging to two different families and three different subfamilies: CYP1A2, CYP2E1, and CYP2C19. In summary, we have demonstrated that MALDI TOF-based peptide mass fingerprinting of different cytochrome P450 isozymes can provide not only qualitative but quantitative data, too.The superfamily of cytochrome(s) P450 (P450) plays a key role in hepatic and extrahepatic drug metabolism, and qualitative and quantitative analyses of the P450 isozyme expression in a particular organ are critical in predicting a metabolic fate of a particular drug or in examination of the potential drug-drug interaction. The number of sequenced and named different P450 isozymes surpassed 3100 (dnelson.utmem.edu/CytochromeP450.html), and the degree of sequence homology, particularly among P450s belonging to the same subfamily, is high (Nelson et al., 1996). None of the existing research approaches to the analysis of individual P450 forms, which include specific P450 inhibitors (Halpert et al., 1994;Kobayashi et al., 2003) or substrates (Kobayashi et al., 2002;Stresser et al., 2002), antibodybased identification (Gelboin et al., 1999;Shou et al., 2000), and mRNA-based analysis (Chow et al., 1999;Zhang et al., 1999), is in a position to provide reliable quantitative and qualitative information on the individual P450 composition in a given type of microsomes. First, only a minority of known P450 isozymes is fully characterized by substrate specificity, and since they exhibit a broad, often overlapping substrate specificity, there is no known substrate or inhibitor that is absolutely specific for an individual P450 isozyme. Second, the high degree of sequence homology among members of the P450 superfamily confounds high specificity of antibody-based analysis, particularly among members of the same subfamily. Third, the application of a quantitative mRNA analysis for the evaluation of P450 isozyme expression...

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Section: Maldi Tof-based Quantitation Of P450ssupporting

confidence: 65%

Section: Resultsmentioning

confidence: 99%

Quantitative Analysis of Cytochrome P450 Isozymes by Means of Unique Isozyme-Specific Tryptic Peptides: A Proteomic Approach

Alterman¹,

Kornilayev²,

Duzhak³

et al. 2005

Drug Metab Dispos

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“…Arachidic acid is a 20-carbon saturated fatty acid present in triacylglycerols from animal fat and some vegetable oils (Hlongwane et al, 2001). We decided to employ this fatty acid in the remainder of the experiments because it produced the highest stimulus for TNF-␣ expression in the hypothalamus and because some previous reports have shown its property to be transported across the blood-brain barrier (Strosznajder et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

Saturated Fatty Acids Produce an Inflammatory Response Predominantly through the Activation of TLR4 Signaling in Hypothalamus: Implications for the Pathogenesis of Obesity

Milanski¹,

Degasperi²,

Coope³

et al. 2009

J. Neurosci.

951

853

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In animal models of diet-induced obesity, the activation of an inflammatory response in the hypothalamus produces molecular and functional resistance to the anorexigenic hormones insulin and leptin. The primary events triggered by dietary fats that ultimately lead to hypothalamic cytokine expression and inflammatory signaling are unknown. Here, we test the hypothesis that dietary fats act through the activation of toll-like receptors 2/4 and endoplasmic reticulum stress to induce cytokine expression in the hypothalamus of rodents. According to our results, long-chain saturated fatty acids activate predominantly toll-like receptor 4 signaling, which determines not only the induction of local cytokine expression but also promotes endoplasmic reticulum stress. Rats fed on a monounsaturated fat-rich diet do not develop hypothalamic leptin resistance, whereas toll-like receptor 4 loss-of-function mutation and immunopharmacological inhibition of toll-like receptor 4 protects mice from diet-induced obesity. Thus, toll-like receptor 4 acts as a predominant molecular target for saturated fatty acids in the hypothalamus, triggering the intracellular signaling network that induces an inflammatory response, and determines the resistance to anorexigenic signals.

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“…Current developments in MALDI hardware [1,2] and increased attention paid to sample preparation have changed the situation. A number of recent publications describe application of MALDI TOF for quantitative studies [3][4][5][6][7][8], including favorable comparisons of MALDI TOF with the more traditional ESI LC/MS quantitation schemes [9 -11]. When installed on QqQ instruments, a MALDI source demonstrated good comparability with an ESI source on the same instrument in quantitative analysis of LMW compounds [12].…”

mentioning

confidence: 99%

MALDI TOF/TOF tandem mass spectrometry as a new tool for amino acid analysis

Gogichaeva

Williams

Alterman

2007

J. Am. Soc. Mass Spectrom.

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This is the first report of an application of collisionally induced fragmentation of amino acids (AA) and their derivatives by MALDI TOF/TOF tandem mass spectrometry (MS). In this work, we collected the data on high-energy fragmentation reactions of a large group of protonated amino acids and their derivatives with the goal of determining which product ions are analyte specific and if yields of these fragment could be used for quantitative analysis. From 34 different amino acids (20 ␣-amino acids, ␤-amino acids, homocysteine, GABA, and modified AA Met sulfone and sulfoxide, hydroxyproline, etc.) we observed that high yields of the target specific immonium ions and fragmentation patterns are most similar to EI or FAB CID on sector instruments. The major exceptions were two highly basic amino acids, Arg and Orn. It is noted that neither ␤-, ␥-, nor ␦-amino acids produce immonium ions. As might be predicted from high-energy CID work on peptides from the sectors and TOF/TOF, the presence of specific indicator ions in MALDI tandem MS allows distinguishing isomeric and isobaric amino acids. These indicator ions, in combination with careful control of data acquisition, ensure quantitative analysis of amino acids. We believe our data provide strong ince their introduction in the late 1980s, the soft-ionization techniques, electrospray ionization (ESI) and matrix-assisted laser desorption/ ionization (MALDI), have became the major ionization methods for the analysis of biological molecules. While analysis of large biopolymers is successfully performed by both techniques, analysis of low molecular weight (LMW) molecules such as amino acids, pharmaceuticals, hormones, etc. is predominantly performed by ESI mass spectrometry with quadrupole mass analyzers. The major reasons not to use MALDI have been the abundance of matrix ions in the low mass region (100 to 600 u) and the lack of reasonable precursor selection in a MALDI tandem instrument. Also, until recently, MALDI was not considered to be a technique capable of providing robust quantitative data. Current developments in MALDI hardware [1,2] and increased attention paid to sample preparation have changed the situation. A number of recent publications describe application of MALDI TOF for quantitative studies [3][4][5][6][7][8], including favorable comparisons of MALDI TOF with the more traditional ESI LC/MS quantitation schemes [9 -11]. When installed on QqQ instruments, a MALDI source demonstrated good comparability with an ESI source on the same instrument in quantitative analysis of LMW compounds [12].Amino acid analysis (AAA) is one of the areas where ESI LC/MS/MS was successfully applied. ESI LC/ MS/MS has become a standard analytical tool in the analysis of low molecular weight physiological or therapeutic molecules of interest [13][14][15][16].We decided to examine if the combination of highenergy collision and prompt ion detection characteristic for MALDI TOF MS/MS could be applied for AAA. The proposed application of MALDI TOF for AAA contains some advantages o...

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Comparative quantitative fatty acid analysis of triacylglycerols using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry and gas chromatography

Cited by 24 publications

References 0 publications

Quantitative Analysis of Cytochrome P450 Isozymes by Means of Unique Isozyme-Specific Tryptic Peptides: A Proteomic Approach

Quantitative Analysis of Cytochrome P450 Isozymes by Means of Unique Isozyme-Specific Tryptic Peptides: A Proteomic Approach

Saturated Fatty Acids Produce an Inflammatory Response Predominantly through the Activation of TLR4 Signaling in Hypothalamus: Implications for the Pathogenesis of Obesity

MALDI TOF/TOF tandem mass spectrometry as a new tool for amino acid analysis

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