Analysis of O-linked reducing oligosaccharides released by an in-line flow system

Reducing O-linked oligosaccharides from bovine submaxillary mucin, bovine fetuin, and porcine gastric mucin were recovered by nonreductive alkaline beta-elimination from an in-line flow system. Glycoproteins where attached to a solid support using hydrophobic interaction with alkali-resistant Poros reversed phase beads and a flow of alkali released the oligosaccharides. The alkali was subsequently neutralized by a continuous flow through cation exchange resin. The released oligosaccharides in the flow were trapped in a cartridge filled with graphitized carbon. Salt-free oligosaccharides could be recovered as a concentrated solution by elution with organic solvents from the cartridge. The glycosylation pattern of the released oligosaccharides was compared with the conventionally released and reduced oligosaccharides recovered from alkaline beta-elimination in the presence of borohydride. In general, the recovery from the in-line release was sometimes lower than from the reductive elimination method, but it was shown that alkaline degradation of reducing oligosaccharides was limited in this system. Liquid chromatography using graphitized carbon packing and high pH mobile phases together with negative ion electrospray mass spectrometry showed that both neutral and acidic reducing oligosaccharides could be analyzed in a single run. Reducing O-linked oligosaccharides could also be recovered in this way from human glycophorin separated by SDS-PAGE. The polyacrylamide was sufficient to retain the glycoprotein in the gel while the flow of alkali released the oligosaccharides. It was also shown that the alkaline conditions for releasing O-linked oligosaccharides from fetuin would partially release some N-linked oligosaccharides, particularly in the presence of reducing agent.     

一种多肽固相合成方法与纯化策略研究

目的：多肽与小分子化学药物相比,具有生物活性高、特异性强、不容易产生耐药性等特点,是目前新型药物研发的重点领域。多肽的合成直接影响到多肽药物的作用机制以及药物效果,因此需要建立一种更加便捷、高效的多肽合成方法。方法：采用Fmoc固相合成法合成多肽HF01,通过比较氨基酸连接的反应体系以及氨基酸脱保护的反应体系,从中确定最优体系。利用乙酰化基团进行肽链末端保护,经肽链剪切制备干燥的粗肽,最后采用高效液相色谱仪与高分辨质谱仪联用对粗肽进行纯化。结果：确定多肽合成的连接和脱保护反应体系,并获得纯度高达98.3%的线性多肽。结论：建立了一种高效、便捷的多肽合成及纯化方法,提高了实验室合成多肽的效率,为多肽类药物的研发提供技术支撑。     

Evaluation of Anti-Inflammatory Effects of Celery Leaf and Stem Extracts in LPS-Induced RAW 264.7 Cells Using Nitric Oxide Assay and LC-MS Based Metabolomics

The present work demonstrated and compared the anti-inflammatory effects of celery leaf (CLE) and stem (CSE) extracts. LC-MS-based metabolomics were an effective approach to achieve the biomarker identification and pathway elucidation associated with the reduction in inflammatory responses. The celery extracts suppressed LPS-induced NO production in RAW 264.7 cells, and CLE was five times more effective than CSE. Distinct differences were revealed between the control and celery-treated samples among the 24 characteristic metabolites that were identified. In celery-treated LPS cells, reversals of intracellular (citrulline, proline, creatine) and extracellular (citrulline, lysine) metabolites revealed that the therapeutic outcomes were closely linked to arginine metabolism. Reversals of metabolites when treated with CLE (aspartate, proline) indicated targeted effects on the TCA and urea cycles, while, in the case of CSE (histidine, glucose), the glycolysis and the pentose phosphate pathways were implicated. Subsequently, apigenin and bergapten in CLE were identified as potential biomarkers mediating the anti-inflammatory response.     

From LC-MS/MS metabolomics profiling of Kanchanara Guggulu to molecular docking and dynamics simulation of quercetin pentaacetate with aldose reductase

Kanchanara Guggulu (KG) is an important traditional medicine that is prescribed by the Ayurveda physicians for the treatment of swellings in various organs such as the thyroid, and lymph nodes. High-resolution mass-spectrometry-based metabolomics found metabolites in KG. LC-MS/MS-based metabolomics analysis of KG identified 2,579 compounds including quercetin and kaempferol derivatives. The molecular docking and dynamics analysis of quercetin pentaacetate with aldose reductase is documented for further consideration in drug discovery.     

Chiral liquid chromatography-tandem mass spectrometric methods for stereoisomeric pharmaceutical determinations

The characterization of the drug metabolism and pharmacokinetic (DMPK) profiles of stereoisomers is a fundamental aspect of the drug discovery and development processes. Therefore, chiral drug bioassays are very important to pharmaceutical and biomedical researchers. The recent developments in chiral liquid chromatography coupled to atmospheric pressure ionization tandem mass spectrometry (LC-API-MS/MS) for the analysis of pharmaceuticals are reviewed. Various ionization techniques including electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric photoionization (APPI) interfaced with chiral liquid chromatographic methods are described in terms of their ionization efficiencies, matrix effects and limitations. Examples were selected to demonstrate the applicability of these methods for enantioselective bioanalysis.     

Mechanism of hydroxyl radical scavenging by rebamipide: identification of mono-hydroxylated rebamipide as a major reaction product

Rebamipide, an antiulcer agent, is known as a potent hydroxyl radical (_•OH) scavenger. In the present study, we further characterized the scavenging effect of rebamipide against _•OH generated by ultraviolet (UV) irradiation of hydrogen peroxide (H₂O₂), and identified the reaction products to elucidate the mechanism of the reaction. Scavenging effect of rebamipide was accessed by ESR using DMPO as a _•OH-trapping agent after UVB exposure (305 nm) to H₂O₂ for 1 min in the presence of rebamipide. The signal intensity of _•OH adduct of DMPO (DMPO-OH) was markedly reduced by rebamipide in a concentration-dependent fashion as well as by dimethyl sulfoxide and glutathione as reference radical scavengers. Their second order rate constant values were 5.62 × 10₁₀, 8.16 × 10₉ and 1.65 × 10₁₀ M_-1 s_-1, respectively. As the rebamipide absorption spectrum disappeared during the reaction, a new spectrum grew due to generation of rather specific reaction product. The reaction product was characterized by LC-MS/MS and NMR measurements. Finally, a hydroxylated rebamipide at the 3-position of the 2(1H)-quinolinone nucleus was newly identified as the major product exclusively formed in the reaction between rebamipide and the _•OH generated by UVB/H₂O₂. Specific formation of this product explained the molecular characteristics of rebamipide as a potential _•OH scavenger.     

Comparison of protein solubilization methods suitable for proteomic analysis of soybean seed proteins

Extraction of soybean seed proteins for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry analysis is challenging and inconsistent. In this study, we compared four different protein extraction/solubilization methods-urea, thiourea/urea, phenol, and a modified trichloroacetic acid (TCA)/acetone-to determine their efficacy in separating soybean seed proteins by 2D-PAGE. In all four methods, seed storage proteins were well separated by 2D-PAGE with minor variations in the intensity of the spots. The thiourea/urea and TCA methods showed higher protein resolution and spot intensity of all proteins compared with the other two methods. In addition, several less abundant and high molecular weight proteins were clearly resolved and strongly detected using the thiourea/urea and TCA methods. Protein spots obtained from the TCA method were subjected to mass spectrometry analysis to test their quality and compatibility. Fifteen protein spots were selected, digested with trypsin, and analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography mass spectrometry (LC-MS). The proteins identified were beta-conglycinin, glycinin, Kunitz trypsin inhibitor, alcohol dehydrogenase, Gly m Bd 28K allergen, and sucrose binding proteins. These results suggest that the thiourea/urea and TCA methods are efficient and reliable methods for 2D separation of soybean seed proteins and subsequent identification by mass spectrometry.     

Protein fragmentation via liquid chromatography-quadrupole time-of-flight mass spectrometry: the use of limited sequence information in structural characterization

Fragmentation and "top-down" sequencing of intact proteins by mass spectrometry (MS) is most commonly performed by infusion of protein solutions into Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. However, the high cost of this instrumentation, coupled with the need to infuse "clean" solutions (lacking standard biological buffers), limits broad application of this technique. The current study describes an alternative approach to top-down sequencing using in-source fragmentation on quadrupole time-of-flight (Q-Tof) instrumentation coupled with reversed-phase liquid chromatography (LC). Application of this technique to purified recombinant samples yielded protein fragments during routine LC-MS analysis. The presence of multiple N- and C-terminal fragments allowed localization of structural modifications without proteolytic digestion. The method was extended to complex samples by using LC conditions that provided high-resolution protein separation. Utility of the method was illustrated by real-time monitoring of protein modifications occurring in reconstituted apoptosomes. These experiments illustrate that intact protein mass and limited sequence information can be obtained simultaneously on an LC timescale. This approach will allow a wide variety of laboratories to routinely apply top-down sequencing to problems in structural characterization, protein purification, and biomarker identification.     

In vitro degradation of the antimicrobial human peptide HEM-gamma 130-146 in plasma analyzed by a validated quantitative LC-MS/MS procedure

In stability studies during preclinical drug development, the human antimicrobial peptide hHEM-gamma 130-146 shows progressive N-terminal degradation in plasma. To determine this effect, we developed and validated a selective and quantitative muHPLC-MS/MS procedure for this compound. Following deproteinization by precipitation, reversed-phase separation is performed with a time-saving two-column design online coupled to an ion trap mass spectrometer for electrospray ionization MS detection. Using a linear calibration curve obtained with synthetic external standards ranging nearly two orders of magnitude, we achieved good precision (repeatability and reproducibility: 5-15%), accuracy (-3 to 15%), and ruggedness with a lower limit of quantification at 0.29 microg/ml plasma (0.15 microM). Because of good linearity (r2>0.999), the recovery (84+/-3%) and ion suppression (86+/-4% remaining intensity) were calculated from specifically prepared calibration curves. The developed procedure was applied to human and animal plasma samples. Incubations in the presence and absence of proteinase inhibitors revealed at least an aminopeptidase M activity for the initial N-terminal truncation of tryptophan (W130) and a putative glutaminyl-peptide cyclotransferase activity for the resulting intermediate starting with the bared glutamine residue (Q131). The calculated periods of half-change demonstrated exceeding interspecies variations, whereas the intraspecies variations were only between 20 and 30%. The current procedure is valuable as a generic method for pharmaceutical purposes, and data give important information for further development toward a potential natural drug candidate.