The pomegranate peel is a by-product of pomegranate fruit rich in polyphenols. In this study, pomegranate peel polyphenols were explored using LC-MS/MS, and punicalagin was the most abundant compound. The highest yield (505.89 ± 1.73 mg/g DW) of punicalagin was obtained by ultrasonic-assisted extraction (UAE) with the ethanol concentration of 53%, sample-to-liquid ratio of 1:25 w/v, ultrasonic power of 757 W, and extraction time of 25 min. Punicalagin was further purified by the macroporous resin D101 and prep-HPLC, reaching the purity of 92.15%. The purified punicalagin had the IC50 of 82 ± 0.02 µg/mL against α-glucosidase, similar to the punicalagin standard with IC50 of 58 ± 0.014 µg/mL, both exhibiting a mixed inhibitory mechanism. Molecular docking further revealed that a steric hindrance with the intermolecular energy of -7.99 kcal/mol was formed between punicalagin and α-glucosidase. Overall, pomegranate peel is a promising source of punicalagin to develop anti-diabetic functional foods.
Tropidolaemus wagleri and Cryptelytrops purpureomaculatus are venomous pit viper species commonly found in Malaysia. Tandem mass spectrometry analysis of the crude venoms has detected different proteins in T. wagleri and C. purpureomaculatus. They were classified into 13 venom protein families consisting of enzymatic and nonenzymatic proteins. Enzymatic families detected in T. wagleri and C. purpureomaculatus venom were snake venom metalloproteinase, phospholipase A₂, ʟ-amino acid oxidase, serine proteases, 5'-nucleotidase, phosphodiesterase, and phospholipase B. In addition, glutaminyl cyclotransferase was detected in C. purpureomaculatus. C-type lectin-like proteins were common nonenzymatic components in both species. Waglerin was present and unique to T. wagleri-it was not in C. purpureomaculatus venom. In contrast, cysteine-rich secretory protein, bradykinin-potentiating peptide, and C-type natriuretic peptide were present in C. purpureomaculatus venom. Composition of the venom proteome of T. wagleri and C. purpureomaculatus provides useful information to guide production of effective antivenom and identification of proteins with potential therapeutic applications.
The lack of specific and accurate therapeutic targets poses a challenge in the treatment of cervical cancer (CC). Global proteomics has the potential to characterize the underlying and intricate molecular mechanisms that drive the identification of therapeutic candidates for CC in an unbiased manner. The present study assessed human papillomavirus (HPV)‑induced proteomic alterations to identify key cancer hallmark pathways and protein‑protein interaction (PPI) networks, which offered the opportunity to evaluate the possibility of using these for targeted therapy in CC. Comparative proteomic profiling of HPV‑transfected (HPV16/18 E7), HPV‑transformed (CaSki and HeLa) and normal human keratinocyte (HaCaT) cells was performed using the liquid chromatography‑tandem mass spectrometry (LC‑MS/MS) technique. Both label‑free quantification and differential expression analysis were performed to assess differentially regulated proteins in HPV‑transformed and ‑transfected cells. The present study demonstrated that protein expression was upregulated in HPV‑transfected cells compared with in HPV‑transformed cells. This was probably due to the ectopic expression of E7 protein in the former cell type, in contrast to its constitutive expression in the latter cell type. Subsequent pathway visualization and network construction demonstrated that the upregulated proteins in HPV16/18 E7‑transfected cells were predominantly associated with a diverse array of cancer hallmarks, including the mTORC1 signaling pathway, MYC targets V1, hypoxia and glycolysis. Among the various proteins present in the cancer hallmark enrichment pathways, phosphoglycerate kinase 1 (PGK1) was present across all pathways. Therefore, PGK1 may be considered as a potential biomarker. PPI analysis demonstrated a direct interaction between p130 and polyubiquitin B, which may lead to the degradation of p130 via the ubiquitin‑proteasome proteolytic pathway. In summary, elucidation of the key signaling pathways in HPV16/18‑transfected and ‑transformed cells may aid in the design of novel therapeutic strategies for clinical application such as targeted therapy and immunotherapy against cervical cancer.
Limited honey production worldwide leads to higher market prices, thus making it prone to adulteration. Therefore, regular physicochemical analysis is imperative for ensuring authenticity and safety. This study describes the physicochemical and antioxidant properties of Apis cerana honey sourced from the islands of Lombok and Bali, showing their unique regional traits. A comparative analysis was conducted on honey samples from Lombok and Bali as well as honey variety from Malaysia. Moisture content was found slightly above 20% in raw honey samples from Lombok and Bali, adhering to the national standard (SNI 8664:2018) of not exceeding 22%. Both honey types displayed pH values within the acceptable range (3.40-6.10), ensuring favorable conditions for long-term storage. However, Lombok honey exhibited higher free acidity (78.5±2.14 meq/kg) than Bali honey (76.0±1.14 meq/kg), surpassing Codex Alimentarius recommendations (≤50 meq/kg). The ash content, reflective of inorganic mineral composition, was notably lower in Lombok (0.21±0.02 g/100) and Bali honey (0.14±0.01 g/100) compared to Tualang honey (1.3±0.02 g/100). Electric conductivity, indicative of mineral content, revealed Lombok and Bali honey with lower but comparable values than Tualang honey. Hydroxymethylfurfural (HMF) concentrations in Lombok (14.4±0.11 mg/kg) and Bali (17.6±0.25 mg/kg) were slightly elevated compared to Tualang honey (6.4±0.11 mg/kg), suggesting potential processing-related changes. Sugar analysis revealed Lombok honey with the highest sucrose content (2.39±0.01g/100g) and Bali honey with the highest total sugar content (75.21±0.11 g/100g). Both honeys exhibited lower glucose than fructose content, aligning with Codex Alimentarius guidelines. The phenolic content, flavonoids, and antioxidant activity were significantly higher in Lombok and Bali honey compared to Tualang honey, suggesting potential health benefits. Further analysis by LC-MS/MS-QTOF targeted analysis identified various flavonoids/flavanols and polyphenolic/phenolic acid compounds in Lombok and Bali honey. The study marks the importance of characterizing the unique composition of honey from different regions, ensuring quality and authenticity in the honey industry.
Multiclass of endocrine disrupting chemicals (EDCs) such as nine perfluoroalkyl and polyfluoroalkyl substances (PFAS), five bisphenols, and four parabens were analysed in tap water samples from Malaysia's Klang Valley region. All samples were analysed using liquid chromatography mass tandem spectrometry (LC-MS/MS) with limit of quantitation (LOQ) ranged between 0.015 and 5 ng/mL. Fifteen of the 18 EDCs were tested positive in tap water samples, with total EDC concentrations ranging from 0.28 to 5516 ng/L for all 61 sampling point locations. In a specific area of the Klang Valley, the total concentration of EDCs was found to be highest in Hulu Langat, followed by Sepang, Putrajaya, Petaling, Kuala Lumpur, Seremban, and Gombak/Klang. PFAS and paraben were the most found EDCs in all tap water samples. Meanwhile, ethyl paraben (EtP) exhibited the highest detection rate, with 90.2% of all locations showing its presence. Over 60% of the regions showed the presence of perfluoro-n-butanoic acid (PFBA), perfluoro-n-hexanoic acid (PFHXA), perfluoro-n-octanoic acid (PFOA), perfluoro-n-nonanoic acid (PFNA), and perfluoro-1-octanesulfonate (PFOS), whereas the frequency of detection for other compounds was less than 40%. The spatial distribution and mean concentrations of EDCs in the Klang Valley regions revealed that Hulu Langat, Petaling Jaya, and Putrajaya exhibited higher levels of bisphenol A (BPA). On the other hand, Kuala Lumpur and Sepang displayed the highest mean concentrations of PFBA. In the worst scenario, the estimated daily intake (EDI) and risk quotient of some EDCs in this study exceeded the acceptable daily limits recommended by international standards, particularly for BPA, PFOA, PFOS, and PFNA, where the risk quotient (RQ) was found to be greater than 1, indicating a high risk to human health. The increasing presence of EDCs in tap water is undoubtedly a cause for concern as these substances can have adverse health consequences. This highlights the necessity for a standardised approach to evaluating EDC exposure and its direct impact on human populations' health.
Affinity purification coupled to mass spectrometry (AP-MS) is a powerful method to analyze protein-protein interactions (PPIs). The AP-MS approach provides an unbiased analysis of the entire protein complex and is useful to identify indirect interactors. However, reliable protein identification from the complex AP-MS experiments requires appropriate control of false identifications and rigorous statistical analysis. Another challenge that can arise from AP-MS analysis is to distinguish bona fide interacting proteins from the non-specifically bound endogenous proteins or the "background contaminants" that co-purified by the bait experiments. In this chapter, we will first describe the protocol for performing in-solution trypsinization for the samples from the AP experiment followed by LC-MS/MS analysis. We will then detail the MaxQuant workflow for protein identification and quantification for the PPI data derived from the AP-MS experiment. Finally, we describe the CRAPome interface to process the data by filtering against contaminant lists, score the interactions and visualize the protein interaction networks.
The general population is exposed to bisphenol A (BPA) orally, parenterally, transdermally, and environmentally as a result of the use of BPA in food packaging, plastics, and personal care products. The majority of the population nowadays (91-99%) has detectable levels of BPA inside their body. In this study, we successfully performed an inexpensive, rapid, and simple protein precipitation procedure for extraction of BPA from human plasma, followed by analysis by LC-MS/MS. This method was specifically developed for handling large numbers of samples with minimum cost and volume of sample. The developed method was accurate, precise, and reproducible for quantification of BPA from human plasma samples in the concentration range of 10-2000 ng/mL. The method was performed on samples from 150 healthy volunteers who were enrolled in the study. The mean of observed BPA level was 2.22 ± 9.91 ng/mL. Higher BPA levels were observed for females compare to that of males (p-value = 0.002), the BPA levels were higher in participants 33 years of age and older compared to those less than 33 years of age (p-value = 0.000), then the BPA levels higher in subjects with tap water as source of drinking (p-value = 0.005). This method may be valuable for general risk assessment of BPA for a large and varied population because of its efficiency and economical aspects.
This study aimed to estimate the Malaysian adult population's current dietary exposure and margin of exposure (MOE) to the carcinogenic processing contaminant, acrylamide. A total of 448 samples from 11 types of processed foods were collected randomly throughout Malaysia in the year 2015 and 2016. Acrylamide was analysed in samples using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) with a limit of detection (LOD) of 10 μg/kg and a limit of quantification (LOQ) of 25 μg/kg. The highest average level of acrylamide (772 ± 752 μg/kg) was found in potato crisps, followed by French fries (415 ± 914 μg/kg) and biscuits (245 ± 195 μg/kg). The total acrylamide exposure for the adult Malaysian was 0.229 and 1.77 μg/kg body weight per day for average and high consumers, respectively. The MOE were 741 and 1875 for the average consumer based on cancer and non-cancer effects of acrylamide, respectively. Meanwhile, for high consumers, the MOE is 96 for cancer and 243 for non-cancer effects. These findings indicate potential carcinogenic risks from acrylamide exposure among Malaysian adults, especially in Malay and other Bumiputra groups compared to Chinese, Indian, and other ethnic groups, while non-cancer effects appeared less concerning.
This work employed the model protein β-lactoglobulin (BLG) to investigate the contribution of microstructural changes to regulating the interaction patterns between protein and flavor compounds through employing computer simulation and multi-spectroscopic techniques. The formation of molten globule (MG) state-like protein during the conformational evolution of BLG, in response to ultrasonic (UC) and heat (HT) treatments, was revealed through multi-spectroscopic characterization. Differential MG structures were distinguished by variations in surface hydrophobicity and the microenvironment of tryptophan residues. Fluorescence quenching measurements indicated that the formation of MG enhanced the binding affinity of heptanal to protein. LC-MS/MS and NMR revealed the covalent bonding between heptanal and BLG formed by Michael addition and Schiff-base reactions, and MG-like BLG exhibited fewer chemical shift residues. Molecular docking and molecular dynamics simulation confirmed the synergistic involvement of hydrophobic interactions and hydrogen bonds in shaping BLG-heptanal complexes thus promoting the stability of BLG structures. These findings indicated that the production of BLG-heptanal complexes was driven synergistically by non-covalent and covalent bonds, and their interaction processes were influenced by processes-induced formation of MG potentially tuning the release and retention behaviors of flavor compounds.
Current in silico modelling techniques, such as molecular dynamics, typically focus on compounds with the highest concentration from chromatographic analyses for bioactivity screening. Consequently, they reduce the need for labour-intensive in vitro studies but limit the utilization of extensive chromatographic data and molecular diversity for compound classification. Compound permeability across the blood-brain barrier (BBB) is a key concern in central nervous system (CNS) drug development, and this limitation can be addressed by applying cheminformatics with codeless machine learning (ML). Among the four models developed in this study, the Random Forest (RF) algorithm with the most robust performance in both internal and external validation was selected for model construction, with an accuracy (ACC) of 87.5% and 86.9% and area under the curve (AUC) of 0.907 and 0.726, respectively. The RF model was deployed to classify 285 compounds detected using liquid chromatography quadrupole time-of-flight mass spectrometry (LCQTOF-MS) in Kelulut honey; of which, 140 compounds were screened with 94 descriptors. Seventeen compounds were predicted to permeate the BBB, revealing their potential as drugs for treating neurodegenerative diseases. Our results highlight the importance of employing ML pattern recognition to identify compounds with neuroprotective potential from the entire pool of chromatographic data.
Bisphenol analogues are prevalent globally because of rampant usage and imprecise processing techniques, prompting alerts about environmental and health hazards. The method employed in this study by solid phase extraction (SPE) and liquid chromatography-tandem quadrupole mass spectrometer (LC-MS/MS) for both quantification and qualitative analysis of the bisphenol compounds in the surface water samples. The coastal and estuarine surface water of Port Dickson and Lukut ranges from 1.32 ng/L to 1890.51 ng/L of bisphenol analogues. BPF mean concentration at 1143.88 ng/L is the highest, followed by BPA and BPS at 59.01 ng/L and 10.96 ng/L, respectively. Based on RQm for bisphenol analogues, the highest for BPF at 2.49 (RQ > 1, high risk), followed by BPS at 0.12 (0.1
Helminth parasites are a group of complex metazoans from various taxonomic families. Excretory secretory (ES) by-products, secreted by living parasites from the surface, appeared to modulate the host immunological response towards helminth infection. This study aims to investigate the effect of ES antigen from helminth parasite on colorectal cell viability. Worm were cultured in phosphate-buffered saline (PBS x1) at 37°C for 24 hours after being rinsed in sterile PBS. Using a mortar and pestle, the worm was crushed vigorously using PBS. The obtained excretory secretory (ES) antigens were extracted and filtered using a 0.22 µM filter and stored at -20°C for further assay. For LCMS, 100 µl of the extract was analysed using Agilent ZORBAX Eclipse Plus C18 Rapid Resolution HT. The extraction of ES antigen (10 µg/ml and 20 µg/ml) was used for cell viability studies using CRC cell line HCT 116. Cell viability and MTT assay were conducted as per the protocol mentioned in the MTT kit. The liquid chromatography and mass spectrometry (LCMS) data indicated that the ES antigen contained metabolic compounds, namely fatty acid, amino alcohol, indoles, sterols, glycosides, and sphingoids. For the Ascaris lumbricoides LCMS analyses, around 405 metabolic peaks were detected. Out of which, 58 were detected via the database were identified, while several compounds detected have anticancer properties. The MTT assay indicated that after 24 hours and 48 hours of exposure, all treated cells showed a decrease in cell viability compared to the control group. The preliminary studies demonstrated that the ES antigen from Ascaris lumbricoides has some ability to decrease the cell viability of the HCT116 CRC cell line. Further studies are needed to examine the cell cycle arrest and apoptosis effect of the ES antigen towards the CRC cell line.
Borassus flabellifer L., commonly known as Asian palmyra, is native to South and Southeast Asia. The endosperms of B. flabellifer (known as nungu in Dravidian culture) are widely consumed during the summer season. It is rich in various nutrients and helps in reducing weight, treating skin and digestive issues, lowering body temperature, and managing migraines and diabetes. This study focuses on identifying the small molecules and proteins from the two varieties of B. flabellifer tender fruit endosperms collected from districts around Chennai, Tamil Nadu, India. The collected free nuclear endosperm was subjected to direct extraction and the mesocarp and cellular endosperms were lyophilized and homogenized. Metabolites were extracted by hexane, methanol, and chloroform and investigated using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). The compounds identified were from the classes of carboxylic acids, flavonoids, amino acids, alkaloids, fatty acids, oligopeptides, vitamins, and glycosides. High-performance liquid chromatography (HPLC) technique was employed to estimate the quantity of amino acids, wherein the total amino acid in the green variety was found to be higher than in the black variety. Proteins were identified after simulating with a gastrointestinal enzyme using liquid chromatography tandem mass spectrometry (LC-MS/MS)-based peptide mass fingerprinting. The different mineral oxides present in the tender fruit endosperm were identified using X-ray diffraction studies, which confirmed the presence of mineral oxides, such as Br1.25ClO2.75Pb3.88, calcium zirconium tantalum oxide, and barium fluoroniobate. This study validates the presence of bioactive metabolites in green and black varieties of B. flabellifer tender fruit endosperm with a range of activities, such as anti-inflammatory, antibacterial, anticancer, and anti-diabetic properties.
Parabens (PBs) are esters of p-hydroxybenzoic acid, and there are growing concerns due to their potential to disrupt endocrine function and their wide use as preservatives in foodstuffs, including beverages. The consumption of bottled and hand-shaken teas is gradually replacing traditional tea consumption through brewing. However, no study has reported PB concentrations in different types of teas or packaging and their associated health risks. Our aim was to determine the concentration of PBs (methyl- (MetPB), ethyl- (EthPB), propyl- (PropPB), butyl-paraben (ButPB)) in green, black, and oolong teas in two varieties of products (bottled and hand-shaken teas), using UPLC-MS/MS. Additionally, we estimated the health risks associated with tea consumption in the general adult population of Taiwan. A Monte Carlo simulation was applied to estimate the distribution of daily PB intake through bottled (n = 79) and hand-shaken (n = 71) tea consumption. Our findings revealed geometric mean concentrations in bottled green/black/oolong teas were 714.1/631.2/532.1 ng/L for MetPB, 95.2/ 30.5/14.9 ng/L for EthPB, 77.9/28.3/non-detected (ND) ng/L for PropPB, and 69.3/26.6/ND ng/L for ButPB. Hand-shaken green/black/oolong teas exhibited concentrations of 867.5/2258/1307 ng/L for MetPB, 28.5/28.8/14.5 ng/L for EthPB, 25.4/18.3/17.8 ng/L for PropPB, and 30.3/18.0/15.5 ng/L for ButPB. The median MetPB concentrations in hand-shaken black (2333 ng/L) and oolong teas (1215 ng/L) were significantly higher than those in bottled black (595.4 ng/L) and oolong teas (489.3 ng/L). Conversely, median concentrations of EthPB, PropPB, and ButPB in bottled teas were significantly higher than those in hand-shaken teas. MetPB was the predominant PB, constituting 73.2-91.9% in bottled teas and 85-94% in hand-shaken teas. Our results showed no health risks associated with bottled or hand-shaken tea consumption based on reference doses. However, the study highlights the importance of continued vigilance given the potential chronic exposure to PBs from various sources, necessitating ongoing concern despite the absence of immediate risks from tea consumption.
A simple liquid chromatography tandem mass spectrometry method was developed and validated according to the guidelines of the US Food and Drug Administration and the European Medicines Agency for a simultaneous quantification of levetiracetam (LEV) and its metabolite, UCB L057 in the plasma of patients. A 0.050 mL plasma sample was prepared by a simple and direct protein precipitation with 0.450 mL acetonitrile (ACN) containing 1 µg/mL of internal standard (IS, diphenhydramine), then vortex mixed and centrifuged. A 0.100 mL of the clear supernatant was diluted with 0.400 mL water and well mixed. A 0.010 mL of the resultant solution was injected into an Agilent Zorbax SB-C18 (2.1 mm×100 mm, 3.5 µm) column with an isocratic elution at 0.5 mL/min using a mixture of 0.1% formic acid in water and ACN (40:60 v/v). Detection was performed using an AB Sciex API 3000 triple quadrupole mass spectrometer, equipped with a Turbo Ion Spray source, operating in a positive mode: LEV at transition 171.1>154.1, UCB L057 at 172.5>126.1, and IS at 256.3>167.3; with an assay run time of 2 minutes. The lower limit of quantification (LLOQ) for both LEV and UCB L057 was validated at 0.5 µg/mL, while their lower limit of detection (LOD) was 0.25 µg/mL. The calibration curves were linear between 0.5 and 100 µg/mL for both analytes. The inaccuracy and imprecision of both intra-assay and inter-assay were less than 10%. Matrix effects were consistent between sources of plasma and the recoveries of all compounds were between 100% and 110%. Stability was established under various storage and processing conditions. The carryovers from both LEV and UCB L057 were less than 6% of the LLOQ and 0.13% of the IS. This assay method has been successfully applied to a population pharmacokinetic study of LEV in patients with epilepsy.
A multidrug-resistant clinical bacteria strain GB11 was isolated from a wound swab on the leg of a patient. Identity of stain GB11 as Pseudomonas aeruginosa was validated by using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Detection of the production of signaling molecules, N-acylhomoserine lactones (AHLs), was conducted using three different bacterial biosensors. A total of four different AHLs were found to be produced by strain GB11, namely N-butyryl homoserine lactone (C4-HSL), N-hexanoylhomoserine lactone (C6-HSL), N-octanoyl homoserine lactone (C8-HSL) and N-3-oxo-dodecanoylhomoserine lactone (3-oxo-C12-HSL) using high resolution liquid chromatography tandem mass spectrometry (LC-MS/MS). Of these detected AHLs, 3-oxo-C12-HSL was found to be the most abundant AHL produced by P. aeruginosa GB11.
Research was carried out to estimate the levels of capsaicin and dihydrocapsaicin that may be found in some heat tolerant chili pepper genotypes and to determine the degree of pungency as well as percentage capsaicin content of each of the analyzed peppers. A sensitive, precise, and specific ultra fast liquid chromatographic (UFLC) system was used for the separation, identification and quantitation of the capsaicinoids and the extraction solvent was acetonitrile. The method validation parameters, including linearity, precision, accuracy and recovery, yielded good results. Thus, the limit of detection was 0.045 µg/kg and 0.151 µg/kg for capsaicin and dihydrocapsaicin, respectively, whereas the limit of quantitation was 0.11 µg/kg and 0.368 µg/kg for capsaicin and dihydrocapsaicin. The calibration graph was linear from 0.05 to 0.50 µg/g for UFLC analysis. The inter- and intra-day precisions (relative standard deviation) were <5.0% for capsaicin and <9.9% for dihydrocapsaicin while the average recoveries obtained were quantitative (89.4%-90.1% for capsaicin, 92.4%-95.2% for dihydrocapsaicin), indicating good accuracy of the UFLC method. AVPP0705, AVPP0506, AVPP0104, AVPP0002, C05573 and AVPP0805 showed the highest concentration of capsaicin (12,776, 5,828, 4,393, 4,760, 3,764 and 4,120 µg/kg) and the highest pungency level, whereas AVPP9703, AVPP0512, AVPP0307, AVPP0803 and AVPP0102 recorded no detection of capsaicin and hence were non-pungent. All chili peppers studied except AVPP9703, AVPP0512, AVPP0307, AVPP0803 and AVPP0102 could serve as potential sources of capsaicin. On the other hand, only genotypes AVPP0506, AVPP0104, AVPP0002, C05573 and AVPP0805 gave a % capsaicin content that falls within the pungency limit that could make them recommendable as potential sources of capsaicin for the pharmaceutical industry.
Mango peel is a good source of protease but remains an industrial waste. This study focuses on the optimization of polyethylene glycol (PEG)/dextran-based aqueous two-phase system (ATPS) to purify serine protease from mango peel. The activity of serine protease in different phase systems was studied and then the possible relationship between the purification variables, namely polyethylene glycol molecular weight (PEG, 4000-12,000 g·mol(-1)), tie line length (-3.42-35.27%), NaCl (-2.5-11.5%) and pH (4.5-10.5) on the enzymatic properties of purified enzyme was investigated. The most significant effect of PEG was on the efficiency of serine protease purification. Also, there was a significant increase in the partition coefficient with the addition of 4.5% of NaCl to the system. This could be due to the high hydrophobicity of serine protease compared to protein contaminates. The optimum conditions to achieve high partition coefficient (84.2) purification factor (14.37) and yield (97.3%) of serine protease were obtained in the presence of 8000 g·mol(-1) of PEG, 17.2% of tie line length and 4.5% of NaCl at pH 7.5. The enzymatic properties of purified serine protease using PEG/dextran ATPS showed that the enzyme could be purified at a high purification factor and yield with easy scale-up and fast processing.
Here a mass spectrometry-based platform for the analysis of glycoproteins is presented. Glycopeptides and released glycans are analyzed, the former by quadrupole orthogonal time-of-flight liquid chromatography/mass spectrometry (QoTOF LC/MS) and the latter by permethylation analysis using matrix-assisted laser desorption/ionization (MALDI)-TOF MS. QoTOF LC/MS analysis reveals the stochastic distribution of glycoforms at occupied sequons, and the latter provides a semiquantitative assessment of overall protein glycosylation. Hydrophilic interaction chromatography (HILIC) was used for unbiased enrichment of glycopeptides and was validated using five model N-glycoproteins bearing a wide array of glycans, including high-mannose, complex, and hybrid subtypes such as sulfo and sialyl forms. Sialyl and especially sulfated glycans are difficult to analyze because these substitutions are labile. The conditions used here allow detection of these compounds quantitatively, intact, and in the context of overall glycosylation. As a test case, we analyzed influenza B/Malaysia/2506/2004 hemagglutinin, a component of the 2006-2007 influenza vaccine. It bears 11 glycosylation sites. Approximately 90% of its glycans are high mannose, and 10% are present as complex and hybrid types, including those with sulfate. The stochastic distribution of glycoforms at glycosylation sites is revealed. This platform should have wide applications to glycoproteins in basic sciences and industry because no apparent bias for any glycoforms is observed.
High temperature liquid chromatography using water-rich and superheated water eluent is evaluated as a new approach for the separation of selected triazole fungicides, hexaconazole, tebuconazole, propiconazole, and difenoconazole. Using a polybutadiene-coated zirconia column at temperatures of 100-150 degrees C, clear separations were achieved when 100% purified water was utilized as organic-free eluent. Excellent limits of detection down to pg level were obtained for the separation of the triazole fungicides under optimum conditions. Van't Hoff plots for the separations were linear suggesting that no changes occurred in the retention mechanism over the temperature range studied.