A method for hydrolyzing and determining the amino acid compositions of picomole quantities of proteins in less than 3 hours

A method is described for quantitatively hydrolyzing proteins in 45 min and for analyzing the hydrolysates by high-performance liquid chromatography in an additional 52 min. The α-amino acids were detected by the fluorescence of their o-phthaldialdehyde derivatives. Ten picomoles of each of the commonly occuring α-amino acids could be reliably determined. The method described yielded OPA-ethanethiolamino acid derivatives that were stable for $\text{1}\text{h}$ h and the HPLC method produced a better separation than previously published methods.     

A method for profiling classes of plant hormones and their metabolites using liquid chromatography-electrospray ionization tandem mass spectrometry: an analysis of hormone regulation of thermodormancy of lettuce (Lactuca sativa L.) seeds

A highly selective and sensitive method for the simultaneous analysis of several plant hormones and their metabolites is described. The method combines high-performance liquid chromatography (HPLC) with positive and negative electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to quantify a broad range of chemically and structurally diverse compounds. The addition of deuterium-labeled analogs for these compounds prior to sample extraction permits accurate quantification by multiple reaction monitoring (MRM). Endogenous levels of abscisic acid (ABA), abscisic acid glucose ester (ABA-GE), 7'-hydroxy-abscisic acid (7'-OH-ABA), phaseic acid (PA), dihydrophaseic acid (DPA), indole-3-acetic acid (IAA), indole-3-aspartate (IAAsp), zeatin (Z), zeatin riboside (ZR), isopentenyladenine (2iP), isopentenyladenosine (IPA), and gibberellins (GA)1, GA3, GA4, and GA7 were determined simultaneously in a single run. Detection limits ranged from 0.682 fmol for Z to 1.53 pmol for ABA. The method was applied to the analysis of plant hormones and hormonal metabolites associated with seed dormancy and germination in lettuce (Lactuca sativa L. cv. Grand Rapids), using extracts from only 50 to 100 mg DW of seed. Thermodormancy was induced by incubating seeds at 33 degrees C instead of 23 degrees C. Germinating seeds transiently accumulated high levels of ABA-GE. In contrast, thermodormant seeds transiently accumulated high levels of DPA after 7 days at 33 degrees C. GA1 and GA3 were detected during germination, and levels of GA1 increased during early post-germinative growth. After several days of incubation, thermodormant seeds exhibited a striking transient accumulation of IAA, which did not occur in seeds germinating at 23 degrees C. We conclude that hormone metabolism in thermodormant seeds is surprisingly active and is significantly different from that of germinating seeds.     

UPLC/MS based method for quantitative determination of fatty acid composition in Gram-negative and Gram-positive bacteria

Quantitative fatty acid composition of microorganisms at various growth space points is required for understanding membrane associated processes of cells, but the majority of the relevant publications still restrict to the relative compositions. In the current study, a simple and reliable method for quantitative measurement of fatty acid content in bacterial biomass without prior derivatization using ultra performance liquid chromatography-electrospray ionization mass spectrometry was developed. The method was applied for investigating the influence of specific growth rate and pH on the fatty acid profiles of two biotechnologically important microorganisms — Gram-negative bacteria Escherichia coli and Gram-positive bacteria Lactococcus lactis grown in controlled physiological states. It was found that the membranes of slowly growing cells are more rigid and that the fatty acid fraction of the cells of L. lactis diminishes considerably with increasing growth rate.     

Serum steroid profiling by isotopic dilution-liquid chromatography-mass spectrometry: comparison with current immunoassays and reference intervals in healthy adults

Background

The simultaneous, rapid and reliable measurement of a wide steroid panel is a powerful tool to unravel physiological and pathological hormone status. Clinical laboratories are currently dominated by high-throughput immunoassays, but these methods lack specificity due to cross-reactivity and matrix interferences. We developed and validated an isotopic dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method for the simultaneous measurement of cortisol, corticosterone, 11deoxycortisol, androstenedione, deoxycorticosterone (DOC), testosterone, 17OHprogesterone, dehydroepiandrosterone (DHEA) and progesterone in serum, and compared it to routine immunoassays employed in our laboratory. We also established adult reference intervals in 416 healthy subjects.

Methods

0.9 ml of serum were spiked with labelled internal standards (IS) and extracted on C18 cartridges. Eluate was injected into a two-dimensional LC-system, purified in a perfusion column and separated on a C8 column during a 21 min gradient run. Analytes were revealed by atmospheric pressure chemical ionization (APCI) followed by multiple reaction monitoring (MRM) analysis.

Results

Of the four immunoassays compared with the ID-LC-MS/MS method, only the results of ElecsysE170 for cortisol, testosterone in males and progesterone > 1 ng/ml were in agreement with ID-LC-MS/MS. ElecsysE170 for testosterone in females and progesterone < 1 ng/ml, Immulite2000 for androstenedione, DSL-9000 for DHEA and 17OHP Bridge for 17OHprogesterone, respectively, showed poor agreement. Reference intervals and steroid age and fertility related fluctuations were established.

Conclusion

Our ID-LC-MS/MS method proved to be reliable and sensitive in revealing steroid circulating concentrations in adults and in highlighting the limits of routine immunoassays at low concentrations.     

Sensitive UHPLC-MS/MS quantification method for 4β- and 4α-hydroxycholesterol in plasma for accurate CYP3A phenotyping

4β-Hydroxycholesterol (4β-OHC) is formed by Cytochrome P450 (CYP)3A and has drawn attention as an endogenous phenotyping probe for CYP3A activity. However, 4β-OHC is also increased by cholesterol autooxidation occurring in vitro due to dysregulated storage and in vivo by oxidative stress or inflammation, independent of CYP3A activity. 4α-hydroxycholesterol (4α-OHC), a stereoisomer of 4β-OHC, is also formed via autooxidation of cholesterol, not by CYP3A, and thus may have clinical potential in reflecting the state of cholesterol autooxidation. In this study, we establish a sensitive method for simultaneous quantification of 4β-OHC and 4α-OHC in human plasma using ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Plasma samples were prepared by saponification, two-step liquid-liquid extraction, and derivatization using picolinic acid. Intense [M+H]+ signals for 4β-OHC and 4α-OHC di-picolinyl esters were monitored using electrospray ionization. The assay fulfilled the requirements of the US Food and Drug Administration guidance for bioanalytical method validation, with a lower limit of quantification of 0.5 ng/ml for both 4β-OHC and 4α-OHC. Apparent recovery rates from human plasma ranged from 88.2% to 101.5% for 4β-OHC, and 91.8% to 114.9% for 4α-OHC. Additionally, matrix effects varied between 86.2% and 117.6% for 4β-OHC and between 89.5% and 116.9% for 4α-OHC. Plasma 4β-OHC and 4α-OHC concentrations in healthy volunteers, stage 3–5 chronic kidney disease (CKD) patients, and stage 5D CKD patients as measured by the validated assay were within the calibration ranges in all samples. We propose this novel quantification method may contribute to accurate evaluation of in vivo CYP3A activity.     

Substrate specificity and products of side-reactions catalyzed by jasmonate:amino acid synthetase (JAR1)

Jasmonate:amino acid synthetase (JAR1) is involved in the function of jasmonic acid (JA) as a plant hormone. It catalyzes the synthesis of several JA-amido conjugates, the most important of which appears to be JA-Ile. Structurally, JAR1 is a member of the firefly luciferase superfamily that comprises enzymes that adenylate various organic acids. This study analyzed the substrate specificity of recombinant JAR1 and determined whether it catalyzes the synthesis of mono- and dinucleoside polyphosphates, which are side-reaction products of many enzymes forming acyl approximately adenylates. Among different oxylipins tested as mixed stereoisomers for substrate activity with JAR1, the highest rate of conversion to Ile-conjugates was observed for (+/-)-JA and 9,10-dihydro-JA, while the rate of conjugation with 12-hydroxy-JA and OPC-4 (3-oxo-2-(2Z-pentenyl)cyclopentane-1-butyric acid) was only about 1-2% that for (+/-)-JA. Of the two stereoisomers of JA, (-)-JA and (+)-JA, rate of synthesis of the former was about 100-fold faster than for (+)-JA. Finally, we have demonstrated that (1) in the presence of ATP, Mg(2+), (-)-JA and tripolyphosphate the ligase produces adenosine 5'-tetraphosphate (p(4)A); (2) addition of isoleucine to that mixture halts the p(4)A synthesis; (3) the enzyme produces neither diadenosine triphosphate (Ap(3)A) nor diadenosine tetraphosphate (Ap(4)A) and (4) Ap(4)A cannot substitute ATP as a source of adenylate in the complete reaction that yields JA-Ile.     

Gamma-linolenic acid alters the composition of mitochondrial membrane subfractions,decreases outer mitochondrial membrane binding of hexokinase and alters carnitine palmitoyltransferase I properties in the Walker 256 rat tumour

Gamma-linolenic acid (GLA) is known to be an inhibitor of Walker 256 tumour growth in vivo and causes changes in both mitochondrial structure and cellular metabolism. The aim of the present study was to investigate in greater detail the changes in energy metabolism and ultrastructure induced by GLA in this tumour model. A diet containing 5.5% GLA, which is sufficient to cause a 45% decrease in tumour growth, was found to almost double the triacylglycerol (TAG) content of the tumour and to increase the quantity of 20:3 n?6, 20:4 n?6, 22:4 n?6 and 22:5 n?6 in the TAG fraction as determined by gas chromatography–mass spectrometry (GCMS) analysis. Morphometric analysis of the tumour by electron microscopy confirmed this increase in TAG content, identifying a doubling of lipid droplet content in the GLA dietary group. The surface density of mitochondrial cristae was reduced, along with a reduction in the number of contact sites (CS) and matrix granules. These three parameters are likely indicators of a reduction in mitochondrial metabolic activity. Measurement of hexokinase activity identified that much of the total hexokinase activity was in the mitochondrially bound form (66.5%) in the control tumour and that GLA caused a decrease in the amount of enzyme in the bound form (39.3%). The fatty acyl chain composition of the tumour mitochondrial subfractions, outer membranes (OM), CSs and inner membranes (IM) was determined by GCMS. All subfractions showed considerable increases in 20:3 n?6 and decreases in 18:1 n?9, 18:2 n?6 and 22:6 n?3, when exposed to GLA diet. These changes were reflected in a large increase in the n?6/n?3 ratio in the GLA OM vs. the control OM, 21.299 vs. 6.747, respectively. The maximal activity of OM carnitine palmitoyltransferase I (CPT I) was found to be decreased by 61.6% in the GLA diet group. This was accompanied by a decrease in malonyl CoA sensitivity and a decrease in affinity for 16:0 CoA substrate. Such changes in CPT I may be the cause of cytoplasmic acyl CoA accumulation seen in this tumour model. These effects, together with previously reported increases in lipid peroxidation, lead to the conclusion that GLA may cause inhibition of tumour cell growth through separate but interlinked pathways, all of which eventually lead to apoptosis and a decrease in tumour development. The influence of mitochondrial OM fatty acyl chain composition upon two important enzymes of energy metabolism, hexokinase and CPT I, both of which have been linked to apoptosis, is of considerable importance for future studies on fatty acid-induced cell death.     

High Sensitivity and Wide Linearity LC-MS/MS Method for Oxylipin Quantification in Multiple Biological Samples

Oxylipins are important biological regulators that have received extensive research attention. Due to the extremely low concentrations, large concentration variations, and high structural similarity of many oxylipins, the quantitative analysis of oxylipins in biological samples is always a great challenge. Here, we developed a liquid chromatography-tandem mass spectrometry-based method with high sensitivity, wide linearity, and acceptable resolution for quantitative profiling of oxylipins in multiple biological samples. A total of 104 oxylipins, some with a high risk of detection crosstalk, were well separated on a 150 mm column over 20 min. The method showed high sensitivity with lower limits of quantitation for 87 oxylipins, reaching 0.05–0.5 pg. Unexpectedly, we found that the linear range for 16, 18, and 17 oxylipins reached 10,000, 20,000, and 40,000 folds, respectively. Due to the high sensitivity, while reducing sample consumption to below half the volume of previous methods, 74, 78, and 59 low-abundance oxylipins, among which some were difficult to detect like lipoxins and resolvins, were well quantified in the tested mouse plasma, mouse liver, and human plasma samples, respectively. Additionally, we determined that analytes with multifarious concentrations of over a 1,000-fold difference could be well quantified simultaneously due to the wide linearity. In conclusion, most likely due to the instrumental advancement, this method effectively improves the quantitative sensitivity and linear range over existing methods, which will facilitate and advance the study of the physiological and pathophysiological functions of oxylipins.     

Natural products as modulators of the nuclear receptors and metabolic sensors LXR,FXR and RXR

Nuclear receptors (NRs) represent attractive targets for the treatment of metabolic syndrome-related diseases. In addition, natural products are an interesting pool of potential ligands since they have been refined under evolutionary pressure to interact with proteins or other biological targets.This review aims to briefly summarize current basic knowledge regarding the liver X (LXR) and farnesoid X receptors (FXR) that form permissive heterodimers with retinoid X receptors (RXR). Natural product-based ligands for these receptors are summarized and the potential of LXR, FXR and RXR as targets in precision medicine is discussed.