Amino Acid Analysis in Physiological Samples by GC-MS with Propyl Chloroformate Derivatization and iTRAQ-LC-MS/MS.

Two mass spectrometry-based methods for the quantitative analysis of free amino acids are described. The first method uses propyl chloroformate/propanol derivatization and gas chromatography-quadrupole mass spectrometry (GC-qMS) analysis in a single-ion monitoring mode. Derivatization is carried out directly in the aqueous samples, thereby allowing automation of the entire procedure, including addition of reagents, extraction, and injection into the GC-MS.

Expression and function of methylthioadenosine phosphorylase in chronic liver disease.

Unlabelled: To study expression and function of methylthioadenosine phosphorylase (MTAP), the rate-limiting enzyme in the methionine and adenine salvage pathway, in chronic liver disease.

Design: MTAP expression was analyzed by qRT-PCR, Western blot and immunohistochemical analysis. Levels of MTA were determined by liquid chromatography-tandem mass spectrometry.

Changes in the hepatic mitochondrial and membrane proteome in mice fed a non-alcoholic steatohepatitis inducing diet.

Non-alcoholic steatohepatitis (NASH) accounts for a large proportion of cryptic cirrhosis in the Western societies. Nevertheless, we lack a deeper understanding of the underlying pathomolecular processes, particularly those preceding hepatic inflammation and fibrosis. In order to gain novel insights into early NASH-development from the first appearance of proteomic alterations to the onset of hepatic inflammation and fibrosis, we conducted a time-course analysis of proteomic changes in liver mitochondria and membrane-enriched fractions of female C57Bl/6N mice fed either a mere steatosis or NASH inducing diet.

Early changes in the liver-soluble proteome from mice fed a nonalcoholic steatohepatitis inducing diet.

Despite the increasing incidence of nonalcoholic steatohepatitis (NASH) with the rise in lifestyle-related diseases such as the metabolic syndrome, little is known about the changes in the liver proteome that precede the onset of inflammation and fibrosis. Here, we investigated early changes in the liver-soluble proteome of female C57BL/6N mice fed an NASH-inducing diet by 2D-DIGE and nano-HPLC-MS/MS. In parallel, histology and measurements of hepatic content of triglycerides, cholesterol and intermediates of the methionine cycle were performed.

Amino acid analysis in physiological samples by GC-MS with propyl chloroformate derivatization and iTRAQ-LC-MS/MS.

Two mass spectrometry-based methods for the quantitative analysis of free amino acids are described. The first method uses propyl chloroformate/propanol derivatization and gas chromatography-quadrupole mass spectrometry (GC-qMS) analysis in single-ion monitoring mode. Derivatization is carried out directly in aqueous samples, thereby allowing automation of the entire procedure, including addition of reagents, extraction, and injection into the GC-MS.

Quantitative profiling of tryptophan metabolites in serum, urine, and cell culture supernatants by liquid chromatography-tandem mass spectrometry.

A sensitive, selective, and comprehensive method for the quantitative determination of tryptophan and 18 of its key metabolites in serum, urine, and cell culture supernatants was developed. The analytes were separated on a C18 silica column by reversed-phase liquid chromatography and detected by electrospray ionization tandem mass spectrometry in positive ion multiple reaction monitoring (MRM) mode, except for indoxyl sulfate which was measured in negative ion MRM mode in a separate run. The limits of detection and lower limits of quantification were in the range of 0.

Down-regulation of methylthioadenosine phosphorylase (MTAP) induces progression of hepatocellular carcinoma via accumulation of 5'-deoxy-5'-methylthioadenosine (MTA).

Recently, we have shown that down-regulation of methylthioadenosine phosphorylase (MTAP) in hepatocellular carcinoma (HCC) cells enhances the invasive potential and the resistance against cytokines. Here, we aimed at investigating the molecular mechanism underlying this tumor-promoting effect and expanded the analysis to a large series of human HCC tissues. Liquid chromatography tandem mass spectrometry revealed that reduced MTAP expression resulted in higher intra- and extracellular concentrations of 5'-deoxy-5'-methylthioadenosine (MTA) in cultivated HCC cells and, concordantly, higher levels of MTA in HCC tissue.

Quantification of intermediates of the methionine and polyamine metabolism by liquid chromatography-tandem mass spectrometry in cultured tumor cells and liver biopsies.

By means of liquid chromatography-tandem mass spectrometry we showed recently, that the chromosomal deletion or inactivation of the methylthioadenosine phosphorylase (MTAP) gene led to the accumulation of 5'-deoxy-5'-(methylthio)adenosine (MTA) in cancer cells. Here, we expanded the method to other key intermediates of the methionine and polyamine pathways to further elucidate the molecular consequences of a lack of MTAP activity. Employing multiple-reaction monitoring, limits of detection and lower limits of quantification in the range of 2.

Direct and tumor microenvironment mediated influences of 5'-deoxy-5'-(methylthio)adenosine on tumor progression of malignant melanoma.

Recent studies have shown that a loss of methylthioadenosine phosphorylase (MTAP) gene expression exerts a tumor-promoting effect, including induction of invasiveness, enhanced cell proliferation, and resistance against cytokines. To date, the molecular mechanisms underlying these effects remain unknown. Since the loss of MTAP expression resulted in induced secretion of 5'-deoxy-5'-(methylthio)adenosine (MTA), we hypothesized that MTA might modulate the observed effects.

Quantitative analysis of 5'-deoxy-5'-methylthioadenosine in melanoma cells by liquid chromatography-stable isotope ratio tandem mass spectrometry.

The frequent deletion of the human chromosomal region 9p21, including the methylthioadenosine phosphorylase (MTAP) gene, is hypothesized to lead to the intra- and/or extracellular accumulation of 5'-deoxy-5'-methylthioadenosine (MTA) in cancer cells and the subsequent promotion of tumor progression. The lack of sensitive methodology for the direct measurement of MTA in tumor cells has hampered the testing of this hypothesis to date. A liquid chromatography electrospray ionization tandem mass spectrometry method (LC-MS/MS) was developed for the absolute quantitative determination of MTA in cell culture media and cell extracts using stable isotope labeled MTA as an internal standard.