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Direct mass spectrometry analysis of biofluid samples using slug-flow microextraction nano-electrospray ionization.

ABSTRACT: Direct mass spectrometry (MS) analysis of biofluids with simple procedures represents a key step in the translation of MS techniques to clinical and point-of-care applications. The current study reports the development of a single-step method using slug-flow microextraction and nano-electrospray ionization for MS analysis of organic compounds in blood and urine. High sensitivity and quantitation precision have been achieved in the analysis of therapeutic and illicit drugs in 5??L samples. Real-time chemical derivatization has been incorporated for analyzing anabolic steroids. The monitoring of enzymatic functions has also been demonstrated with cholinesterase in wet blood. The reported study encourages the future development of disposable cartridges, which function with simple operation to replace the traditional complex laboratory procedures for MS analysis of biological samples.

SUBMITTER: Ren Y

PROVIDER: S-EPMC4469778 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Direct mass spectrometry analysis of biofluid samples using slug-flow microextraction nano-electrospray ionization.

Ren Yue Y McLuckey Morgan N MN Liu Jiangjiang J Ouyang Zheng Z

Angewandte Chemie (International ed. in English) 20141005 51

Direct mass spectrometry (MS) analysis of biofluids with simple procedures represents a key step in the translation of MS techniques to clinical and point-of-care applications. The current study reports the development of a single-step method using slug-flow microextraction and nano-electrospray ionization for MS analysis of organic compounds in blood and urine. High sensitivity and quantitation precision have been achieved in the analysis of therapeutic and illicit drugs in 5 μL samples. Real-t ...[more]

PMID: 25284028

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Project description:We present the first comprehensive capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS) analysis of post-translational modifications derived from H1 and core histones. Using a capillary electrophoresis system equipped with a sheathless high-sensitivity porous sprayer and nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-ESI-MS) as two complementary techniques, we characterized H1 histones isolated from rat testis. Without any pre-separation of the perchloric acid extraction, a total of 70 different modified peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e, and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-MS only, and 11 solely with LC-ESI-MS. Immobilized metal-affinity chromatography enrichment prior to MS analysis yielded a total of 55 phosphopeptides; 22 of these peptides could be identified only by CESI-MS, and 19 only by LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1 modification sites, including 31 phosphorylation sites, of which 8 were novel sites. For the analysis of core histones, we chose a different strategy. In a first step, the sulfuric-acid-extracted core histones were pre-separated using reverse-phase high-performance liquid chromatography. Individual rat testis core histone fractions obtained in this way were digested and analyzed via bottom-up CESI-MS. This approach yielded the identification of 42 different modification sites including acetylation (lysine and N(α)-terminal); mono-, di-, and trimethylation; and phosphorylation. When we applied CESI-MS for the analysis of intact core histone subtypes from butyrate-treated mouse tumor cells, we were able to rapidly detect their degree of modification, and we found this method very useful for the separation of isobaric trimethyl and acetyl modifications. Taken together, our results highlight the need for additional techniques for the comprehensive analysis of post-translational modifications. CESI-MS is a promising new proteomics tool as demonstrated by this, the first comprehensive analysis of histone modifications, using rat testis as an example.

Project description:BackgroundWax esters are highly hydrophobic neutral lipids that are major constituents of the cutin and suberin layer. Moreover they have favorable properties as a commodity for industrial applications. Through transgenic expression of wax ester biosynthetic genes in oilseed crops, it is possible to achieve high level accumulation of defined wax ester compositions within the seed oil to provide a sustainable source for such high value lipids. The fatty alcohol moiety of the wax esters is formed from plant-endogenous acyl-CoAs by the action of fatty acyl reductases (FAR). In a second step the fatty alcohol is condensed with acyl-CoA by a wax synthase (WS) to form a wax ester. In order to evaluate the specificity of wax ester biosynthesis, analytical methods are needed that provide detailed wax ester profiles from complex lipid extracts.ResultsWe present a direct infusion ESI-tandem MS method that allows the semi-quantitative determination of wax ester compositions from complex lipid mixtures covering 784 even chain molecular species. The definition of calibration prototype groups that combine wax esters according to their fragmentation behavior enables fast quantitative analysis by applying multiple reaction monitoring. This provides a tool to analyze wax layer composition or determine whether seeds accumulate a desired wax ester profile. Besides the profiling method, we provide general information on wax ester analysis by the systematic definition of wax ester prototypes according to their collision-induced dissociation spectra. We applied the developed method for wax ester profiling of the well characterized jojoba seed oil and compared the profile with wax ester-accumulating Arabidopsis thaliana expressing the wax ester biosynthetic genes MaFAR and ScWS.ConclusionsWe developed a fast profiling method for wax ester analysis on the molecular species level. This method is suitable to screen large numbers of transgenic plants as well as other wax ester samples like cuticular lipid extracts to gain an overview on the molecular species composition. We confirm previous results from APCI-MS and GC-MS analysis, which showed that fragmentation patterns are highly dependent on the double bond distribution between the fatty alcohol and the fatty acid part of the wax ester.

Dataset Information

Direct mass spectrometry analysis of biofluid samples using slug-flow microextraction nano-electrospray ionization.

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Direct mass spectrometry analysis of biofluid samples using slug-flow microextraction nano-electrospray ionization.

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