Shengyuan Xiao

Optimization and quality assessment of the post-digestion 18O labeling based on urea for protein denaturation by HPLC/ESI-TOF mass spectrometry.

The post-digestion (18)O labeling method decouples protein digestion and peptide labeling. This method allows labeling conditions to be optimized separately and increases labeling efficiency. A common method for protein denaturation in proteomics is the use of urea. Though some previous studies have used urea-based protein denaturation before post-digestion (18)O labeling, the optimal (18)O labeling conditions in this case have not been yet reported. Present study investigated the effects of urea concentration and pH on the labeling efficiency and obtained an optimized protocol. It was demonstrated that urea inhibited (18)O incorporation depending on concentration. However, a urea concentration between 1 and 2M had minimal effects on labeling. It was also demonstrated that the use of FA to quench the digestion reaction severely affected the labeling efficiency. This study revealed the reason why previous studies gave different optimal pH for labeling. They neglect the effects of different digestion conditions on the labeling conditions. Excellent labeling quality was obtained at the optimized conditions using urea 1-2 M and pH 4.5, 98.4+/-1.9% for a standard protein mixture and 97.2+/-6.2% for a complex biological sample. For a 1:1 mixture analysis of the (16)O- and (18)O-labeled peptides from the same protein sample, the average abundance ratios reached 1.05+/-0.31, demonstrating a good quantitation quality at the optimized conditions. This work will benefit other researchers who pair urea-based protein denaturation with a post-digestion (18)O labeling method.

LC-MS method for determining the activity of semicarbazide-sensitive amine oxidase in rodents

Semicarbazide-sensitive amine oxidase (SSAO) is present in various mammalian tissues and in blood plasma. Elevation of SSAO activity is linked to vascular disorders associated with pathological conditions such as diabetic complications, heart failure and vascular dementia. In the present paper, a high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) method is developed to determine the SSAO activity. Methylamine is used as physiological substrate for the enzyme activity assay of SSAO. Formaldehyde, the enzymatic reaction product, was derivatized by dopamine, and separated by a silica-based pentafluorophenyl column. The calibration curve was linear over the range of 0.03–4.00 μM of formaldehyde concentration, with 0.03 μM the lower limit of quantification (LOQ). The inter-day and intra-day precisions ranged from 2.2% to 7.9% and 4.4% to 9.2% respectively for each quality control sample of formaldehyde at 0.06, 0.50, and 2.00 μM. The accuracy ranged from 96.0% to 111.5%. The limit of detection (LOD) for serum SSAO activity was 1 nmol h−1 mg−1 protein. The method was successfully applied for the determination of SSAO activity in both mouse serum and rat tissues. SSAO activity of the serum in diabetes mice was significantly increased compared with the control and was inhibited by 2-bromoethylamine in vivo. The levels of SSAO activity in rat lung, spleen, aorta, kidney and brain tissues were significantly decreased by semicarbazide in vitro.

Simultaneous Determination of Glycyrrhizin and 15 Flavonoids in Licorice and Blood by High Performance Liquid Chromatography with Ultraviolet Detector

Licorice is the most frequently used herb in traditional Chinese medicine (TCM) with versatile functions. It is also a popular natural dietary supplement. While the dosages is very important for there are some side effects caused by licorice. The composition of licorice, its products should be well determined thereof. A simple method for simultaneous determining sixteen compounds in vary high dynamic range of content has been established. This method based on the detection at the characteristic ultraviolet spectra of different types of compounds in licorice. Glycyrrhizin and fifteen flavonoids were well measured. All of these compounds can be precisely quantified at their characteristic wavelengths. This method has been successfully applied to the analyses of different licorices, Sini Tang decoction, and rat plasma after oral administration of Sini Tang decoction. These compounds were found to be over 3000 times in content (from 0.01 g/g to 34.5 g/g) in some samples.

Potential Toxicity of Chronic Creatine Supplementation in Mice

Creatine is alleged to be a popular nutrition to enhance sports performance. It can be metabolized to methylamine, which is further converted to formaldehyde, hydrogen peroxide and ammonium by semicarbazied-sensitive amine oxidases (SSAO). Until now, there is no scientific available data for demonstration the long-term health risks of chronic creatine ingestion. In this study, we demonstrated that after chronic oral administration of creatine to mice, the level of methylamine and formaldehyde in urine were increased, SSAO activity of serum and tissue were increased simultaneously. SSAO inhibitor (2-bromoethylamine hydrobromide) could substantially increase the methylamine level and decrease the formaldehyde content respectively. A novel high-performance liquid chromatography (HPLC) and electrospray ionization (ESI) mass spectrometry method for the determination of SSAO activity was developed for the first time. Potential toxicity after chronic creatine administration was discussed according to the increase of toxic products content and SSAO activity. To our knowledge, this is the first report on the research of relationship between SSAO and creatine.

Methodology for Determining the Quantity of Major Components in Herbal Complex by LC/MS

Abstract Objective This article aims to study the total elements of Chinese complex, YL2000, by LC-MS. Methods The total flavonoids, total alkaloids, and total coumarins in YL2000 are determined based on the chemical structure (analogue) and using the standard compounds as a reference. Results The quantity of the three elements is similar to the quantity produced by UV test. Conclusions This is the first attempt to determine the total quantity of the elements in herbal complex, which is helpful for the element quality control in Chinese medical complex.

A Study of Cross-Linkage of Formaldehyde with Human Serum Albumin

Its well known that irreversible cross-linked complexes between proteins, produced by formaldehyde interacting with free amino or amide groups to form Schiffs base, are formed in diabetes and aging patients. To identify such specific modified peptides as biomarkers for clinical diagnose of diabetes and AD, cross-linkage of formaldehyde with Human Serum Albumin (HSA) was analyzed by LC/ESI/MS in this study. Eighteen peptides as important biomarkers were identified and the corresponding possible structures of their cross-linkages were also analyzed. Five of those peptides were modified by the reaction of one molecular proportion of formaldehyde with one molecular proportion of amino group. Reaction of one molecular proportion of formaldehyde with two molecular proportions of amino groups were found in 4 of the 18 peptides. In the rest 9 peptides, the structures of the cross- linkages of formaldehyde with amino groups was more complex. Furthermore, several lysine and arginine residues of those peptides were proved as modification sites in HSA cross-linked with formaldehyde.

Determination of S-Catechol-O-Methyltransferase Activity in Rat Tissues Using High-Performance Liquid Chromatography with Electrochemical Detection

A high-performance liquid chromatography with electrochemical detection (HPLC-ECD) method was developed to determine the activity of S-catechol-O-methyltransferase (COMT) in rat tissues including the liver, kidney, lung ,spleen and brain. The 3,4-dihydroxybenzoic acid was used as the substrate for SCOMT in rat tissues. After incubated with different tissues, the product 4-hydroxy-3-methoxybenzoic acid was formed from the substrate and separated to measure the COMT activity. The analysis was performed on a Agilent C8 column (5μm, 150×4.6mm) at 550mV potential. The mobile phase was a mixture of methanol and 0.1MNa2HPO4(pH=3.3) containing 0.13mM ethylenediamine tetraacetic acid (v/v, 14/86) with a flow rate of 1ml/min. This approach was used to study S-COMT activity in vitro from various tissues.

A Novel Method to Enhance Sensitivity in HPLC Detection

The peak compression effect induced by velocity difference has been theoretically and experimentally established. L-tryptophan and 2-nitrophenol were used as model compounds. The peak compression of L-tryptophan and 2-nitrophenol was obtained by the difference of its migration velocity along the column induced by the adjustment of composition of mobile phase. A compression of analytes peak and reduced tailing was observed. Under optimum compression condition, 17-fold and 5.9-fold improvement in the detection sensitivity was obtained compared to elution under non-compressed conditions for L-tryptophan and 2-nitrophenol, respectively.

LC-MS Analysis of Markers for Advanced Glycation Endprocducts -Protein/Peptides Adducts

Advanced glycation end products (AGEs) are a heterogeneous group of proteins that have been modified with glucose or carbohydrates adducts, and are thought to be responsible for many complications of diabetes and aging. Free amino groups of proteins react slowly with reducing sugars such as glucose by the glycation or Maillard reaction, initially forming Schiff bases, which undergo rearrangement to form the relatively stable Amadori products. The Amadori products subsequently degrade into a-dicarbonyl compounds that react with amino groups of proteins to form cross-links, stable end products called advanced glycation end products (AGEs). AGEs have been shown to react (cross-link) with the amino group of the N-terminal amino acid residue and the side-chains of arginine and lysine residues. Therefore, glucose has been shown to be responsible, at least in part, for protein cross-linkage, oxidative stress and cytotoxicity. In this in vitro study model, the cross-linking of glucose (0.05, 0.5, 1.0 and 2.5 M) with bovine serum albumin incubated for different intervals of time (0, 7,14, 21, 28 and 35 days) was studied using LC-MS and Mascot database to search for the possible markers for glucose-peptides adducts. A total of twenty-seven peptides were matched in the database search query for control BSA out of which seven (m/z 922.69, 464.36, 582.41, 681.95, 834.49, 847.59 and 812.87) were found to be the possible markers for AGEs-protein/peptides adducts.

A study on modification of Human Albumin by proteomic technique

Advanced glycation end products (AGEs) are a heterogeneous group of complex compounds which may play a role in the pathogenesis of chronic complications associated with diabetes and aging. Glucose has the ability to crosslink proteins through creation of AGEs. This study was carried out on glycated human albumin (HA) by glucose which were analyzed by LC/MS with the aim of identifying specific peptides from glycated HA. Our objective was to find typical peptides as biomarker for clinical diagnose of diabetes and aging. This method was in vitro incubation of HA with glucose of different concentrations. Glycated and unglycated HA were digested by trypsin. AGE-peptides originated by enzymatic digestion were analysed by LC/MS. Analysis of the LC/MS data show that there were differences between peptides of glycated and unglycated HA. Some peptides detected in unglycated HA cannot be found in glycated HA. These typical peptides were considered as important markers for glycated HA.