Dataset Information

Mass-spectrometric profiling of porphyrins in complex biological samples with fundamental, toxicological, and pharmacological applications.

ABSTRACT: Rapid, high-throughput, and quantitative evaluations of biological metabolites in complex milieu are increasingly required for biochemical, toxicological, pharmacological, and environmental analyses. They are also essential for the development, testing, and improvement of new commercial chemical products. We demonstrate the application of ultra-high performance liquid chromatography-mass spectrometry (uHPLC-MS), employing an electrospray ionization source and a high accuracy quadrupole time-of-flight mass analyzer, for the identification and quantification of a series of porphyrin derivatives in liver: a matrix of particular relevance in toxicological or pharmacological testing. Exact mass is used to identify and quantify the metabolites. Chromatography enhances sensitivity and alleviates potential saturation issues by fanning out the contents of a complex sample before their injection into the spectrometer, but is not strictly necessary for the analysis. Extraction and sample treatment procedures are evaluated and matrix effects discussed. Using this method, the known mechanism of action of a well-characterized porphyrinogenic agent was verified in liver extracts from treated rats. The method was also validated for use with bacterial cells. This exact-mass method uses workhorse instruments available in many laboratories, providing a highly flexible alternative to existing HPLC- and MS/MS-based approaches for the simultaneous analysis of multiple compounds in biological media.

SUBMITTER: Sullivan SA

PROVIDER: S-EPMC4410076 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Mass-spectrometric profiling of porphyrins in complex biological samples with fundamental, toxicological, and pharmacological applications.

Sullivan Sarah A SA Streit Bennett R BR Ferguson Ethan L EL Jean Paul A PA McNett Debra A DA Llames Louis T LT DuBois Jennifer L JL

Analytical biochemistry 20150310

Rapid, high-throughput, and quantitative evaluations of biological metabolites in complex milieu are increasingly required for biochemical, toxicological, pharmacological, and environmental analyses. They are also essential for the development, testing, and improvement of new commercial chemical products. We demonstrate the application of ultra-high performance liquid chromatography-mass spectrometry (uHPLC-MS), employing an electrospray ionization source and a high accuracy quadrupole time-of-f ...[more]

PMID: 25769421

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Project description:Various biological processes at the cellular level are regulated by glycosylation which is a highly microheterogeneous post-translational modification (PTM) on proteins and lipids. The dynamic nature of glycosylation can be studied through metabolic incorporation of non-natural sugars into glycan epitopes and their detection using bio-orthogonal probes. However, this approach possesses a significant drawback due to nonspecific background reactions and ambiguity of non-natural sugar metabolism. Here, we report a probe-free strategy for their direct detection by glycoproteomics and glycomics using mass spectrometry (MS). The method dramatically simplifies the detection of non-natural functional group bearing monosaccharides installed through promiscuous sialic acid, N-acetyl-d-galactosamine (GalNAc) and N-acetyl-d-glucosamine (GlcNAc) biosynthetic pathways. Multistage enrichment of glycoproteins by cellular fractionation, subsequent ZIC-HILIC (zwitterionic-hydrophilic interaction chromatography) based glycopeptide enrichment, and a spectral enrichment algorithm for the MS data processing enabled direct detection of non-natural monosaccharides that are incorporated at low abundance on the N/O-glycopeptides along with their natural counterparts. Our approach allowed the detection of both natural and non-natural sugar bearing glycopeptides, N- and O-glycopeptides, differentiation of non-natural monosaccharide types on the glycans and also their incorporation efficiency through quantitation. Through this, we could deduce interconversion of monosaccharides during their processing through glycan salvage pathway and subsequent incorporation into glycan chains. The study of glycosylation dynamics through this method can be conducted in high throughput, as few sample processing steps are involved, enabling understanding of glycosylation dynamics under various external stimuli and thereby could bolster the use of metabolic glycan engineering in glycosylation functional studies.

Dataset Information

Mass-spectrometric profiling of porphyrins in complex biological samples with fundamental, toxicological, and pharmacological applications.

Publications

Mass-spectrometric profiling of porphyrins in complex biological samples with fundamental, toxicological, and pharmacological applications.

Similar Datasets

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