Lipidomics, the mass spectrometry-based comprehensive analysis of lipids, has attracted attention as an analytical approach to provide novel insight into lipid metabolism and to search for ...biomarkers. However, an ideal method for both comprehensive and quantitative analysis of lipids has not been fully developed. Here, we have proposed a practical methodology for widely targeted quantitative lipidome analysis using supercritical fluid chromatography fast-scanning triple-quadrupole mass spectrometry (SFC/QqQMS) and theoretically calculated a comprehensive lipid multiple reaction monitoring (MRM) library. Lipid classes can be separated by SFC with a normal-phase diethylamine-bonded silica column with high resolution, high throughput, and good repeatability. Structural isomers of phospholipids can be monitored by mass spectrometric separation with fatty acyl-based MRM transitions. SFC/QqQMS analysis with an internal standard-dilution method offers quantitative information for both lipid class and individual lipid molecular species in the same lipid class. Additionally, data acquired using this method has advantages, including reduction of misidentification and acceleration of data analysis. Using the SFC/QqQMS system, alteration of plasma lipid levels in myocardial infarction-prone rabbits to the supplementation of EPA was first observed. Our developed SFC/QqQMS method represents a potentially useful tool for in-depth studies focused on complex lipid metabolism and biomarker discovery.—Takeda, H., Y. Izumi, M. Takahashi, T. Paxton, S. Tamura, T. Koike, Y. Yu, N. Kato, K. Nagase, M. Shiomi, and T. Bamba.
Diabetic kidney disease is a major cause of renal failure that urgently necessitates a breakthrough in disease management. Here we show using untargeted metabolomics that levels of phenyl sulfate, a ...gut microbiota-derived metabolite, increase with the progression of diabetes in rats overexpressing human uremic toxin transporter SLCO4C1 in the kidney, and are decreased in rats with limited proteinuria. In experimental models of diabetes, phenyl sulfate administration induces albuminuria and podocyte damage. In a diabetic patient cohort, phenyl sulfate levels significantly correlate with basal and predicted 2-year progression of albuminuria in patients with microalbuminuria. Inhibition of tyrosine phenol-lyase, a bacterial enzyme responsible for the synthesis of phenol from dietary tyrosine before it is metabolized into phenyl sulfate in the liver, reduces albuminuria in diabetic mice. Together, our results suggest that phenyl sulfate contributes to albuminuria and could be used as a disease marker and future therapeutic target in diabetic kidney disease.
Consideration of the special problems encountered in ultra‐high sensitivity biopolymer sequencing studies has led to the development of a novel quadrupole/orthogonal‐acceleration time‐of‐flight ...tandem mass spectrometer described for the first time here. The performance characteristics of this new geometry are demonstrated, including fully resolved daughter‐ion spectra with mass accuracies of 0.1 dalton, which allow removal of interpretation ambiguities and easy differentiation of charge states even in weak collisionally‐activated decomposition tandem mass spectra. The instrument has been applied to a variety of biopolymer research problems, including the structure determination of major histocompatibility complex peptide antigens using liquid chromatography/electrospray mass spectrometry and nanoflow‐electrospray tandem mass spectrometry, and sequencing capability in the low‐femtomole and attomole ranges is demonstrated.
The plant hormone abscisic acid (ABA) and the jasmonic acid related-compound 12-oxo-phytodienoic acid (OPDA) play crucial roles in seed development, dormancy, and germination. However, a lack of ...suitable techniques for visualising plant hormones has restricted the investigation of their biological mechanisms. In the present study, desorption electrospray ionisation-imaging mass spectrometry (DESI-IMS), a powerful tool for visualising metabolites in biological tissues, was used to visualise ABA and OPDA in immature Phaseolus vulgaris L. seed sections. The mass spectra, peak values and chemical formulae obtained from the analysis of seed sections were consistent with those determined for ABA and OPDA standards, as were the precursor and major fragment ions observed in tandem mass spectrometry (MS/MS) imaging. Furthermore, the precursor and fragment ion images showed similar distribution patterns. In addition, the localisation of ABA and OPDA using DESI-IMS was confirmed using liquid chromatography-MS/MS (LC-MS/MS). The results indicated that ABA was mainly distributed in the radical and cotyledon of the embryo, whereas OPDA was distributed exclusively in external structures, such as the hilum and seed coat. The present study is the first to report the visualisation of plant hormones using IMS, and demonstrates that DESI-IMS is a promising technique for future plant hormone research.
Ultra-high-sensitivity, biopolymer sequencing is a goal in many fields of molecular biology, and collisionally activated decomposition electrospray mass spectrometry (CAD ES MS/MS) using a triple ...quadrupole mass spectrometer has become a method of choice for work in the high- to mid-femtomole range. However, when the detection of ions becomes statistical, as it may in that range, the mass assignment of fragment ions is inaccurate and either sequencing becomes impossible or ambiguities result due, for example, to the closeness in amino acid residue masses (I/L, N or K/Q, E). Some ambiguities may be resolved by synthesizing possible sequences, but this is unsatisfactory. In considering the limitations of triple quadrupole MS/MS with respect to scanning ion detection, resolution, transmission, and mass accuracy, we reasoned that a novel geometry quadrupole orthogonal acceleration time-of-flight (Q-TOF) instrument would have special merit for ultra-high-sensitivity MS/MS sequencing, and suggested its construction for this purpose some three years ago. A prototype Q-TOF has now been built by Micromass Morris et al. (1996), Rapid Commun. Mass Spectrom. 10, 889-896, and in the first research on the instrument, including MHC antigen and filarial nematode glycoprotein studies, we demonstrate low-femtomole- and attomole-range sequencing with mass accuracy of better than 0.1 Da throughout the daughter-ion spectrum, thus removing sequencing ambiguities in some of the most challenging work demanding the highest sensitivity.
Skp1 is a ubiquitous eukaryotic protein found in several cytoplasmic and nuclear protein complexes, including the SCF-type
E3 ubiquitin ligase. In Dictyostelium , Skp1 is hydroxylated at proline 143, ...which is then modified by a pentasaccharide chain. The enzyme activity that attaches
the first sugar, GlcNAc, was previously shown to copurify with the GnT51 polypeptide whose gene has now been cloned using
a proteomics approach based on a quadrupole/time-of-flight hybrid mass spectrometer. When expressed in Escherichia coli , recombinant GnT51 exhibits UDP-GlcNAc:hydroxyproline Skp1 GlcNAc-transferase activity. Based on amino acid sequence alignments,
GnT51 defines a new family of microbial polypeptide glycosyltransferases that appear to be distantly related to the catalytic
domain of mucin-type UDP-GalNAc:Ser/Thr polypeptide α-GalNAc-transferases expressed in the Golgi compartment of animal cells.
This relationship is supported by the effects of site-directed mutagenesis of GnT51 amino acids associated with its predicted
D X D-like motif, DAH. In contrast, GnT51 lacks the N-terminal signal anchor sequence present in the Golgi enzymes, consistent
with the cytoplasmic localization of the Skp1 acceptor substrate and the biochemical properties of the enzyme. The first glycosylation
step of Dictyostelium Skp1 is concluded to be mechanistically similar to that of animal mucin type O -linked glycosylation, except that it occurs in the cytoplasm rather than the Golgi compartment of the cell.
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