Liquid Chromatography-tandem MS Research Articles

Identification and Activity Study of an Impurity Band Observed in the nrSDS-PAGE of Aflibercept.

Aflibercept is a biopharmaceutical targeting vascular endothelial growth factor (VEGF) that has shown promise in the treatment of neovascular age-related macular degeneration (nAMD) and diabetic macular edema (DME) in adults. Quality control studies of aflibercept employing non-reduced SDS-PAGE (nrSDS-PAGE) have shown that a significant variant band (IM1) is consistently present below the main band. Considering the quality control strategy of biopharmaceuticals, structural elucidation and functional studies are required. In this study, the variant bands in nrSDS-PAGE were collected through electroelution and identified by peptide mass fingerprinting based on liquid chromatography-tandem MS (LC-MS/MS). This variant was expressed using knob-into-hole (KIH) design transient transfection for the detection of ligand affinity, binding activity and biological activity. The variant band was formed by C-terminal truncation at position N99 of one chain in the aflibercept homodimer. Then, this variant was successfully expressed using KIH design transient transfection. The ligand affinity of the IM1 truncated variant was reduced by 18-fold, and neither binding activity nor biological activity were detected. The efficacy of aflibercept is influenced by the loss of biological activity of the variant. Therefore, this study supports the development of a quality control strategy for aflibercept.

Small-Scale Serial Size Exclusion Chromatography (s3SEC) for High Sensitivity Top-Down Proteomics of Large Proteoforms.

Top-down-mass spectrometry (MS)-based proteomics has emerged as a premier technology to examine proteins at the proteoform level, enabling characterization of genetic mutations, alternative splicing, and post-translational modifications. However, significant challenges that remain in top-down proteomics include the analysis of large proteoforms and the sensitivity required to examine proteoforms from minimal amounts of sample. To address these challenges, we have developed a new method termed "small-scale serial Size Exclusion Chromatography" (s3SEC), which incorporates a small-scale protein extraction (1 mg of tissue) and serial SEC without postfractionation sample handling, coupled with online high sensitivity capillary reversed-phase liquid chromatography tandem MS (RPLC-MS/MS) for analysis of large proteoforms. The s3SEC-RPLC-MS/MS method significantly enhanced the sensitivity and reduced the proteome complexity across the fractions, enabling the detection of high MW proteoforms previously undetected in one-dimensional (1D)-RPLC analysis. Importantly, we observed a drastic improvement in the signal intensity of high MW proteoforms in early fractions when using the s3SEC-RPLC method. Moreover, we demonstrate that this s3SEC-RPLC-MS/MS method also allows the analysis of lower MW proteoforms in subsequent fractions without significant alteration in proteoform abundance and equivalent or improved fragmentation efficiency to that of the 1D-RPLC approach. Although this study focuses on the use of cardiac tissue, the s3SEC-RPLC-MS/MS method could be broadly applicable to other systems with limited sample inputs.

Concurrent determination of cyanide and thiocyanate in human and swine antemortem and postmortem blood by high-performance liquid chromatography-tandem mass spectrometry.

Cyanide (in the form of cyanide anion (CN-) or hydrogen cyanide (HCN), inclusively represented as CN) can be a rapidly acting and deadly poison, but it is also a common chemical component of a variety of natural and anthropogenic substances. The main mechanism of acute CN toxicity is based on blocking terminal electron transfer by inhibiting cytochrome c oxidase, resulting in cellular hypoxia, cytotoxic anoxia, and potential death. Due to the well-established link between blood CN concentrations and the manifestation of symptoms, the determination of blood concentration of CN, along with the major metabolite, thiocyanate (SCN-), is critical. Because currently there is no method of analysis available for the simultaneous detection of CN and SCN- from blood, a sensitive method for the simultaneous analysis of CN and SCN- from human ante- and postmortem blood via liquid chromatography-tandem MS analysis was developed. For this method, sample preparation for CN involved active microdiffusion with subsequent chemical modification using naphthalene-2,3-dicarboxaldehyde (NDA) and taurine (i.e., the capture solution). Preparation for SCN- was accomplished via protein precipitation and monobromobimane (MBB) modification. The method produced good sensitivity for CN with antemortem limit of detection (LODs) of 219 nM and 605 nM for CN and SCN-, respectively, and postmortem LODs of 352 nM and 509 nM. The dynamic ranges of the method were 5-500µM and 10-500µM in ante- and postmortem blood, respectively. In addition, the method produced good accuracy (100 ± 15%) and precision (≤ 15.2% relative standard deviation). The method was able to detect elevated levels of CN and SCN- in both antemortem (N = 5) and postmortem (N = 4) blood samples from CN-exposed swine compared to nonexposed swine.

Spatially Resolved Proteomics Reveals Lens Suture-Related Cell-Cell Junctional Protein Distributions.

Lens transparency relies on the precise organization of lens fiber cells. The formation of the highly ordered lens architecture results from not only cell-cell adhesion along the lateral interfaces, but also from proper organization of fiber cells tips at lens sutures. Little is known about the cell adhesion between fiber tips at the sutures. The purpose of this study is to map suture-specific protein distributions. Tissue sections were obtained from fresh frozen bovine lenses and washes were performed to remove soluble proteins and to retain membrane and membrane associated proteins. Imaging mass spectrometry (IMS) combined with on-tissue trypsin digestion was used to visualize protein spatial distributions. Sutures and adjacent regions were captured by laser capture microdissection and samples were digested by trypsin. Proteins were analyzed by liquid chromatography tandem MS and quantified by label-free quantification. Protein spatial distributions were confirmed by immunofluorescence. IMS results showed enrichment of adherens junction proteins cadherin-2 and armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF) in both anterior and posterior sutures of bovine lenses. Liquid chromatography tandem MS confirmed higher expression of cadherin-2 and ARVCF and other adherens junction proteins including catenin α2 (CTNNA2) and catenin β1 (CTNNB1) in sutures. In contrast, IMS indicated low expression of gap junction protein connexin 50 and connexin 46 in the suture regions. The localization of cadherin-2 and connexin 50 was confirmed by immunofluorescence. The complementary expression of adherens junction proteins and gap junction proteins in lens suture regions implicates adherens junctions in fiber cell tip adhesion and in maintaining the integrity of the lens.

Characterization of the improved functionality in soybean protein-proanthocyanidins conjugates prepared by the alkali treatment

This study investigated the changes in functionality, structure, and protein composition in soybean protein-proanthocyanidins (SPI-PC) conjugates prepared by the alkali method with dose-ranging PC molecular grafting (0.5, 1, 2 mg/mL). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that more >180 kDa polymers were formed in SPI-PC conjugates with the increase of PC levels. The structural analysis showed that conjugation to PC altered the molecular structure of SPI, appearing the protein-unfolding with PC level-dependent. Liquid chromatography tandem MS analysis (LC/MS-MS) demonstrated that the composition of protein and peptides, as well as allergen and epitopes, were changed after SPI was conjugated with PC. The antioxidant activity of SPI-PC conjugates was increased with the increase of PC levels because high PC contents caused more hydroxyl groups in polyphenols to introduce into the SPI. Emulsifying properties were increased with the increase of PC levels, probably resulting from the unfolding of proteins, increase of random coil and decrease of surface hydrophobicity. Western-bolt and enzyme-linked immunosorbent assay (ELISA) showed a reduced IgE binding capacity in SPI-PC conjugates with PC level-dependent, which was attributed to the reduction of major soybean allergens and the masking or destruction of epitopes induced by structural changes. However, the foaming properties varied from PC levels. Overall, this study demonstrated that SPI-PC conjugates formed using the alkali method have a potential application as hypoallergenic soybean products or functional ingredients in foods. In future studies, suitable reactions (e.g., PC levels) are required to be investigated in SPI-PC conjugates for balancing various functional properties. • PC increased emulsifying, foaming and antioxidant properties in SPI. • The IgE binding capacity in SPI was reduced with the increase in PC levels. • Protein modifications and structural changes in SPI varied from PC levels. • Suitable PC levels are required to balance various functionality in conjugates.

Multi-spectral and proteomic insights into the impact of proanthocyanidins on IgE binding capacity and functionality in soy 11S protein during alkali-heating treatment

This study evaluated the impact of proanthocyanidins on immunoglobulin E (IgE) binding capacity, antioxidant, foaming and emulsifying properties in soy 11S protein following alkali treatment at 80 °C for 20 min. The formation of >180 kDa polymer was observed in the combined heating and proanthocyanidins-conjugation treatment sample (11S-80PC) rather than in the heating treated sample (11S-80) using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The structural analyzes demonstrated that 11S-80PC exhibited more protein unfolding than 11S-80. Heatmap analysis revealed that 11S-80PC had more alteration of peptide and epitope profiles in 11S than in 11S-80. Molecular docking showed that PC could well react with soy protein 11S. Liquid chromatography tandem MS analysis (LC/MS-MS) demonstrated that there was a 35.6 % increase in 11S-80, but a 14.5 % decrease in 11S-80PC for the abundance of total linear epitopes. As a result, 11S-80PC exhibited more reduction in IgE binding capacities than 11S-80 owing to more obscuring and disruption of linear and conformational epitopes induced by structural changes. Moreover, 11S-80PC exhibited higher antioxidant capacities, foaming properties and emulsifying activity than 11S-80. Therefore, the addition of proanthocyanidins could decrease allergenic activity and enhance the functional properties of the heated soy 11S protein.

Plasma Choline Concentration Was Not Increased After a 6-Month Egg Intervention in 6–9-Month-Old Malawian Children: Results from a Randomized Controlled Trial

ABSTRACTBackgroundEggs are a rich source of choline, an essential nutrient important for child growth and development. In a randomized trial of 1 egg/d in young children in Ecuador, an egg intervention led to significant improvements in growth, which were partially mediated by increased plasma choline concentration. A similar trial in Malawi (clinicaltrials.gov: NCT03385252) found little improvement in child growth or development.ObjectivesWe aimed to evaluate the effect of 1 egg/d for 6 mo on plasma choline concentrations in Malawian children enrolled in a randomized trial.MethodsInfants aged 6–9 mo in rural Malawi were randomly assigned to receive 1 egg/d (n = 331) or serve as a nonintervention control (n = 329) for 6 mo. Anthropometric, developmental, and dietary data were collected at baseline and 6-mo follow-up, along with a blood draw. Plasma choline, betaine, dimethylglycine, trimethylamine N-oxide (TMAO), and DHA were measured at both time points using ultrahigh performance liquid chromatography–tandem MS (n = 200 per group). Linear regression analysis was used to determine the difference in plasma choline and related metabolites between groups after 6 mo of intervention.ResultsPlasma choline, betaine, dimethylglycine, and DHA concentrations did not differ between groups at 6-mo follow-up. Plasma TMAO was significantly (26%; 95% CI: 7%, 48%) higher in the egg intervention group in a fully adjusted model.ConclusionsProvision of 1 egg/d for 6 mo did not result in increases in plasma choline or related metabolites, except TMAO. This could partially explain the lack of effect on growth and development. Additional interventions are needed to improve choline status, growth, and development in this population.

Identification of synthetic cannabinoid methyl 2-{[1-(cyclohexylmethyl)-1H-indol-3-yl] formamido}-3-methylbutanoate using modern mass spectrometry and nuclear magnetic resonance techniques

Abstract The samples of plant material suspected to contain new psychoactive substances are very often the subject of chemical-toxicological analyses. Gas chromatography-mass spectrometry (MS), liquid chromatography-quadrupole time-of-flight-MS, and liquid chromatography-tandem MS were applied with the aim to identify synthetic cannabinoid, methyl 2-{[1-(cyclohexylmethyl)-1H-indol-3-yl] formamido}-3-methylbutanoate (MMB-CHMICA) without the analytical standard, which is very often the case when a new drug arrives. The structure of compound was also confirmed by one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy and conformational analysis. After identification, methanolic extract of plant material containing MMB-CHMICA was successfully used for developing a multiple reaction monitoring method on liquid chromatography-tandem MS instrument. The optimization procedure is shown in detail. The complete fragmentation pattern and also the optimization of the extraction procedure of MMB-CHMICA from plant material were shown. The obtained data are useful for forensic, toxicological, and clinical purposes.

Enteral nutrition ameliorates the symptoms of Crohn's disease in mice via activating special pro-resolving mediators through innate lymphoid cells.

Crohn's disease activates the inflammatory reactions to induce intestinal disorders. Enteral nutrition (EN) could exert general immunomodulatory effects. Cecal ligation and perforation (CLP) surgery was utilized to establish Crohn's disease mice models. Survival analysis, hematoxylin-eosin staining, flow cytometry, ELISA, Western blot and liquid chromatography-tandem MS were applied. Baicalein was added to inhibit lipoxygenases. The survival rate was restored and inflammatory injury, exudate neutrophils in peritoneal lavage and serum levels of IL-6 and TNF-α were ameliorated by EN treatment as compared with CLP treatment. EN also increased ILC-3 content, 5/15-LOX level and RvD1-RvD5 in peritoneal lavage. Baicalein reversed all the detected effects of EN except ILC-3 content. EN could activate special pro-resolving mediators (SPMs) through ILCs to mitigate injuries of Crohn's disease.

Correlation between Bone Disease and Genotype in Patients with Type 1 Gaucher Disease. Data from the Spanish Gaucher Disease Registry

Bone involvement is the most common cause of functional limitation, disability and poor quality of life in patients with lysosomal storage disease (LSD). In type 1 Gaucher disease (GD1) intraosseous lesions have a vascular involvement with obstruction and / or local hemorrhage, once established it, secondary irreversible sequels with structure changes are developed. The intimate mechanisms that induce these feared complications are only partially known and the risk factors are not well established. There has been speculation about the relationship between the type of genetic defect and the severity of the disease. The most frequent mutations found in the Spanish population are point direction changes in the GBA gene (c.1226A> G (N370S) and c.1448T> C (L444P). However, other types of mutations (nonsense, splicing, frameshift stop codon, deletions, insertions, recombination) that induce a lower residual enzyme activity and, consequently, a higher storage rate and therefore more intensity of symptoms are found.Objective: To analyze the relationship between different missense mutation and the presence and developing of vascular bone lesions in GD 1 patients.Patients and Methods: We have analyzed the data obtained in our LSD Unit over the last 20 years in the evaluation of bone disease by magnetic resonance imaging with semi-quantitative estimation of the involvement by S-MRI and BMB scores and determination of bone mineral density by ultrasound in 131 GD patients (Andrade-Campos et al 2016). Here we present a sub-study in 85 patients heterozygous for c.1226A> G comparing the data obtained related to their genetic characteristics. According to the genotype were classified the patients in three groups, A: c.1226A> G / c.1448T> C (34), B: c.1226A> G in heterozygosity with other missense mutation (34) and C: c.1226A> G mutation with other types (17). In addition to search for plasma biomarkers associated to bone disease and verify them we have applied a targeted proteomics strategy. We have transformed monocytes to osteoclasts from these groups of patients and by proteomics profiles we have compared with osteoclasts of healthy controls by isobaric tag for relative and absolute quantification (iTRAQ). Labelling with iTRAQ in combination with sample pre-fractionation and nano liquid chromatography-tandem MS was used for identification and quantification of the basal proteome. More than 700 proteins were identified with this strategy. To verify the quantification of the proteins identified we have selected the MRM (multiple reaction monitoring) strategy for differential quantification according their highly sensitive, reproducible and accurate.Results: The mean age at diagnosis for each of the groups was similar. A: 30.85 (6-68)> 30 years: 19 (55.88%), C: 28.52 (4-56)> 30 years: 8 (47%) And significantly lower in B: 18.32 (3-60;> 30years: 10 (29.41%) (p = 0.01). The M/F ratio was balanced in group A (F: 52.9%) while there was predominance of females in group C (64.72%) and males in group B (64.70%). The proportion of splenectomized patients in groups A and B was similar (32.3%), whereas in group C was significantly higher (52.9%) (p = 0.01). Patients in groups A and B presented similar scores S-MRI and BMB 11,2 / 7,32 while group C showed a significant higher score 14.3 / 8.23 (p: 0.01) and higher incidence of osteopenia and osteoporosis. Only 17% of patients in group C and 20% in group B showed uncomplicated bone marrow infiltration compared to 50% in group A. The mean concentration of the biomarkers (chitotriosidase activity and CCL18/PARC) was similar between the three groups. The results of iTRAQ study has identified some target proteins with potential use as biomarkers that will be present in the meeting if the work is selected to be presented.In conclusion, patients with genotype that include an allele with complex mutations, present greater risk of suffering vascular bone complications (osteonecrosis, infarctions, bone demineralization). It is important to consider these genetic characteristics to establish recommendations about the importance of diagnosis and early treatment as well as to avoid splenectomy and to follow up by annual MRI and DEXA. The standard biomarkers of the disease do not establish differences between risk groups. The protein expression analyzed has show differential expression of some proteins that need to be addressed by target proteomic strategy. DisclosuresNo relevant conflicts of interest to declare.

The Isorhamnetin-Containing Fraction of Philippine Honey Produced by the Stingless Bee Tetragonula biroi Is an Antibiotic against Multidrug-Resistant Staphylococcus aureus.

Honey exhibits antibacterial and antioxidant activities that are ascribed to its diverse secondary metabolites. In the Philippines, the antibacterial and antioxidant activities, as well as the bioactive metabolite contents of the honey, have not been thoroughly described. In this report, we investigated the in vitro antibacterial and antioxidant activities of honey from Apis mellifera and Tetragonula biroi, identified the compound responsible for the antibacterial activity, and compared the observed bioactivities and metabolite profiles to that of Manuka honey, which is recognized for its antibacterial and antioxidant properties. The secondary metabolite contents of honey were extracted using a nonionic polymeric resin followed by antibacterial and antioxidant assays, and then spectroscopic analyses of the phenolic and flavonoid contents. Results showed that honey extracts produced by T. biroi exhibits antibiotic activity against Staphylococcal pathogens as well as high antioxidant activity, which are correlated to its high flavonoid and phenolic content as compared to honey produced by A. mellifera. The bioassay-guided fractionation paired with Liquid Chromatography Mass Spectrometry (LCMS) and tandem MS analyses found the presence of the flavonoid isorhamnetin (3-methylquercetin) in T. biroi honey extract, which was demonstrated as one of the compounds with inhibitory activity against multidrug-resistant Staphylococcus aureus ATCC BAA-44. Our findings suggest that Philippine honey produced by T. biroi is a potential nutraceutical that possesses antibiotic and antioxidant activities.

A Rat Model of Orthopedic Implant-Associated Infection for Identification of Staphylococcal Biofilm Proteins.

Secreted bacterial proteins are difficult to identify directly from an infection site due to a limited amount of bacteria and presence of a large quantity of host proteins. Here we describe a rat model of orthopedic implant that allows us to harvest bacterial biofilm materials sufficient for identification of bacterial proteins in the biofilm matrix by liquid chromatography-tandem MS (GeLC-MS/MS) analysis.

Mutation-independent Proteomic Signatures of Pathological Progression in Murine Models of Duchenne Muscular Dystrophy

The absence of the dystrophin protein in Duchenne muscular dystrophy (DMD) results in myofiber fragility and a plethora of downstream secondary pathologies. Although a variety of experimental therapies are in development, achieving effective treatments for DMD remains exceptionally challenging, not least because the pathological consequences of dystrophin loss are incompletely understood. Here we have performed proteome profiling in tibialis anterior muscles from two murine DMD models (mdx and mdx52) at three ages (8, 16, and 80 weeks of age), all n = 3. High-resolution isoelectric focusing liquid chromatography-tandem MS (HiRIEF-LC-MS/MS) was used to quantify the expression of 4974 proteins across all 27 samples. The two dystrophic models were found to be highly similar, whereas multiple proteins were differentially expressed relative to WT (C57BL/6) controls at each age. Furthermore, 1795 proteins were differentially expressed when samples were pooled across ages and dystrophic strains. These included numerous proteins associated with the extracellular matrix and muscle function that have not been reported previously. Pathway analysis revealed multiple perturbed pathways and predicted upstream regulators, which together are indicative of cross-talk between inflammatory, metabolic, and muscle growth pathways (e.g. TNF, INFγ, NF-κB, SIRT1, AMPK, PGC-1α, PPARs, ILK, and AKT/PI3K). Upregulation of CAV3, MVP and PAK1 protein expression was validated in dystrophic muscle by Western blot. Furthermore, MVP was upregulated during, but not required for, the differentiation of C2C12 myoblasts suggesting that this protein may affect muscle regeneration. This study provides novel insights into mutation-independent proteomic signatures characteristic of the dystrophic phenotype and its progression with aging.

Glucocerebrosidases catalyze a transgalactosylation reaction that yields a newly-identified brain sterol metabolite, galactosylated cholesterol

β-Glucocerebrosidase (GBA) hydrolyzes glucosylceramide (GlcCer) to generate ceramide. Previously, we demonstrated that lysosomal GBA1 and nonlysosomal GBA2 possess not only GlcCer hydrolase activity, but also transglucosylation activity to transfer the glucose residue from GlcCer to cholesterol to form β-cholesterylglucoside (β-GlcChol) in vitro β-GlcChol is a member of sterylglycosides present in diverse species. How GBA1 and GBA2 mediate β-GlcChol metabolism in the brain is unknown. Here, we purified and characterized sterylglycosides from rodent and fish brains. Although glucose is thought to be the sole carbohydrate component of sterylglycosides in vertebrates, structural analysis of rat brain sterylglycosides revealed the presence of galactosylated cholesterol (β-GalChol), in addition to β-GlcChol. Analyses of brain tissues from GBA2-deficient mice and GBA1- and/or GBA2-deficient Japanese rice fish (Oryzias latipes) revealed that GBA1 and GBA2 are responsible for β-GlcChol degradation and formation, respectively, and that both GBA1 and GBA2 are responsible for β-GalChol formation. Liquid chromatography-tandem MS revealed that β-GlcChol and β-GalChol are present throughout development from embryo to adult in the mouse brain. We found that β-GalChol expression depends on galactosylceramide (GalCer), and developmental onset of β-GalChol biosynthesis appeared to be during myelination. We also found that β-GlcChol and β-GalChol are secreted from neurons and glial cells in association with exosomes. In vitro enzyme assays confirmed that GBA1 and GBA2 have transgalactosylation activity to transfer the galactose residue from GalCer to cholesterol to form β-GalChol. This is the first report of the existence of β-GalChol in vertebrates and how β-GlcChol and β-GalChol are formed in the brain.

The Use of LipidIMMS Analyzer for Lipid Identification in Ion Mobility-Mass Spectrometry-Based Untargeted Lipidomics.

Untargeted lipidomics aims to comprehensively measure and characterize all lipid species in biological systems. Ion mobility-mass spectrometry (IM-MS) has showed a great potential for untargeted lipidomic analysis. Coupling with liquid chromatography and data-independent tandem MS techniques, acquired IM-MS data set contains four-dimensional information for lipid identification, including m/z of MS1 ion, retention time (RT), collision cross section (CCS), and MS/MS spectra. In this protocol, we introduced a data processing workflow using an integrative web server, namely, LipidIMMS Analyzer, to support accurate lipid identification. The protocol demonstrated the integration of all four dimensional information to achieve unambiguous identifications of lipids in complex biological samples.

Salivary proteomics of canine oral tumors using MALDI-TOF mass spectrometry and LC-tandem mass spectrometry.

Canine oral tumors are relatively common neoplasms in dogs. For disease monitoring and early diagnosis, salivary biomarkers are appropriate because saliva collection is non-invasive and requires no professional skills. In the era of omics, matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF MS) coupled with liquid chromatography-tandem MS (LC-MS/MS) are suitable to identify potential disease-associated peptides and proteins. The present study aimed to use MALDI-TOF MS and LC-MS/MS to search for particular peptide mass fingerprints (PMFs) and conceivable biomarkers in saliva of dogs with early- and late-stage oral melanoma (EOM and LOM, respectively), oral squamous cell carcinoma (OSCC), benign oral tumors (BN), and periodontitis and healthy controls (CP). Pooled saliva samples in each group were used to be representative of population change. Unique PMFs were obtained and specific peptide fragments were sequenced by LC-MS/MS and BLAST-searched with mammalian protein databases. Seven peptide fragments appeared in the tumor groups (EOM, LOM, OSCC and BN) at 1096, 1208, 1322, 1794, 1864, 2354 and 2483 Da, two peptide fragments appeared in the LOM and OSCC groups at 2450 and 3492 Da, and in the CP controls at 2544 and 3026 Da. Also, protein–chemotherapy drug interaction networks were exhibited. Using western blot analysis, the expression of sentrin-specific protease 7 (SENP7), a peptide fragment at 1096 Da, in OSCC was significantly increased, as was the expression of TLR4, a peptide fragment at 3492 Da, in LOM and OSCC, compared with the CP group. The expression of nuclear factor kappa B (NF-κB), a TLR4 partner, was notably increased in OSCC compared with CP, BN and EOM. The expression was also enhanced in LOM compared with EOM. Expressed protein sequences from western blots were verified by LC-MS/MS. Western blots were then performed with individual samples in each group. The results showed the elevated expression of TLR4 in LOM and OSCC, compared with that in CP and BN, the increased expression of NF-κB in LOM and OSCC, compared with CP and in LOM compared with BN, and the enhanced expression of SENP7 in LOM and OSCC, compared with that in CP and BN. In conclusion, discrete clusters of EOM, LOM, OSCC, BN and CP groups and potential protein candidates associated with the diseases were demonstrated by salivary proteomics. Western blot analysis verified SENP7, TLR4 and NF-κB as potential salivary biomarkers of canine oral tumors.

Oxidative modification of skin lipids by cold atmospheric plasma (CAP): A standardizable approach using RP-LC/MS2 and DI-ESI/MS2

Cold atmospheric plasma (CAP) is an emerging source for the locally defined delivery of reactive species, and its clinical potential has been identified in the control of inflammatory processes, such as acute and chronic wounds, or cancerous lesions. Lipids, due to their localization and chemical structure as ideal targets for oxidative species, are relevant modifiers of physiological processes. Human forehead lipids collected on a target were treated by an argon plasma jet and immediately analyzed by direct-infusion high-resolution tandem mass spectrometry (DI-MS2) or liquid chromatography-tandem MS (RP-LC/MS2). Subsequent data analysis was performed by LipidHunter (University of Leipzig), LipidXplorer (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden), and LipidSearch (Thermo Scientific). With either MS method, all major lipid classes of sebum lipids were detected. Significant differences regarding triacylglycerols (predominantly identified in RP-LC/MS2) and ceramides (predominantly identified in DI-MS2) indicate experimental- or approach-inherent distinctions. A CAP-driven oxidation of triacyclglycerols, ceramides, and cholesteryl esters was detected such as truncations and hydroperoxylations, but at a significantly lower extent than expected. Scavenging of reactive species due to naturally present antioxidants in the samples and the absence of a liquid interphase to allow reactive species deposition by the CAP will have contributed to the limited amount of oxidation products observed. In addition, limitations of the software’s capability of identifying unexpected oxidized lipids potentially led to an underestimation of the CAP impact on skin lipids, indicating a need for further software development. With respect to the clinical application of CAP, the result indicates that intact skin with its sebum/epidermal lipid overlay is well protected and that moderate treatment will yield limited (if any) functional consequences in the dermal tissue.

Oxylipin concentration, but not fatty acid composition, is altered in human donor milk pasteurised using both thermal and non-thermal techniques.

Human donor milk (DM) is Holder pasteurised (62·5°C, 30 min) to ensure its microbiological safety for infant consumption. In low-resource settings, flash heating is used to pasteurise milk. Although there is considerable interest in non-thermal alternatives (high hydrostatic pressure processing (HHP) and UVC irradiation) for pasteurisation, their effect on the fatty acid composition is not well understood. Of particular interest is the effect of pasteurisation on the generation of oxylipins. DM from eight mothers containing bacteria >5 × 107 colony-forming units/l was used. In a paired design, each pool of milk underwent four pasteurisation techniques: Holder; flash heating; UVC (250 nm, 25 min) and HHP (500 MPa, 8 min). Fatty acids were quantified by GC-flame ionisation detection and oxylipins derived from arachidonic acid; 18-carbon PUFA (α-linolenic acid, linoleic acid and γ-linolenic acid) and EPA/DHA were measured by liquid chromatography-tandem MS in aliquots of raw and processed milk. There were no significant changes to the composition of fatty acids following all pasteurisation techniques compared with raw milk. The n-6:n-3 ratio remained constant ranging from 6·4 to 6·6. Several arachidonic acid-derived oxylipins were highest post-UVC and elevated post-HHP compared with raw milk. Several oxylipins derived from 18-carbon PUFA (linoleic and α-linolenic acids) were elevated in UVC-treated milk. EPA/DHA-derived oxylipins were on average, unaffected by pasteurisation. Although some PUFA-derived oxylipins were increased following UVC and HHP, no method affected the fatty acid composition of human DM. Further research is needed to determine if varying levels of oxylipins in human DM as a result of processing can potentially mediate cellular signalling; proliferation and apoptosis, especially important for preterm infant development.

MON-188 Characterization and Categorization Based on Genotype-Biochemical Phenotype Association in Fructose-1,6-Bisphosphatase Deficiency

Fructose-1,6-bisphosphatase (FBPase) deficiency, caused by an FBP1 mutation, is an autosomal recessive disorder characterized by hypoglycemia and metabolic acidosis. Due to the rarity of FBPase deficiency, elucidating the mechanism by which the mutations cause enzyme activity loss is challenging. We performed whole-exome sequencing in an adult patient with severe hypoglycemic lactic acidosis and identified that the patient carried compound heterozygous missense mutations of FBP1 (NM_000507.3) with c.491G>A (p.G164D) and c.581T>C (p.F194S). Biochemical analysis using FBP1-KO HepG2 cells generated by CRISPR/Cas9 system revealed that FBP1 mutant (G164D or F194S) overexpression decreased protein expression and enzyme activity loss. The interactome analysis based on Liquid chromatography-tandem MS data for binding partners demonstrated that FBP1, particularly its mutant, interacts with the proteins involved in the molecular chaperone related to unfolded protein response including heat shock protein (HSP). Indeed, G164D and F194S mutants exhibited increased HSPs interaction and aggregated in the endoplasmic reticulum, suggesting involvement of protein misfolding in its pathogenesis. The biochemical phenotypes of all FBP1 missense mutations previously reported were examined and categorized into three functional phenotypes: Type 1 mutations, located at pivotal residues in enzyme activity motifs with no effects on protein expression and intracellular localization; Type 2 mutations, which mediate changes in amino acid hydrophobicity and structurally cluster around the substrate binding pocket, are associated with high HSP-binding, aggregation in the endoplasmic reticulum, and decreased protein expression; and Type 3 mutations, which are likely non-pathogenic mutations without biochemical phenotype. Thus, our findings demonstrate that protein misfolding contributes to FBPase deficiency pathogenesis, particularly in Type 2 mutations; therefore, its missense mutations can be classified into three categories based on genotype functional phenotype association.

Prevalence and predictors of vitamin D deficiency in a nationally representative sample of adults participating in the 2011-2013 Australian Health Survey.

Vitamin D deficiency is recognised as a public health problem globally, and a high prevalence of deficiency has previously been reported in Australia. This study details the prevalence of vitamin D deficiency in a nationally representative sample of Australian adults aged ≥25 years, using an internationally standardised method to measure serum 25-hydroxyvitamin D (25(OH)D) concentrations and identifies demographic and lifestyle factors associated with vitamin D deficiency. We used data from the 2011-2013 Australian Health Survey (n 5034 with complete information on potential predictors and serum 25(OH)D concentrations). Serum 25(OH)D concentrations were measured by a liquid chromatography-tandem MS that is certified to the reference measurement procedures developed by the National Institute of Standards and Technology, Ghent University and the US Centers for Disease Control and Prevention. Vitamin D deficiency and insufficiency were defined as serum 25(OH)D concentrations <50 nmol/l and 50 to <75 nmol/l, respectively. Overall, 20 % of participants (19 % men; 21 % women) were classified as vitamin D deficient, with a further 43 % classified as insufficient (45 % men; 42 % women). Independent predictors of vitamin D deficiency included being born in a country other than Australia or the main English-speaking countries, residing in southern (higher latitude) states of Australia, being assessed during winter or spring, being obese, smoking (women only), having low physical activity levels and not taking vitamin D or Ca supplements. Given our increasingly indoor lifestyles, there is a need to develop and promote strategies to maintain adequate vitamin D status through safe sun exposure and dietary approaches.