A generalized covariate-adjusted top-scoring pair algorithm with applications to diabetic kidney disease stage classification in the Chronic Renal Insufficiency Cohort (CRIC) Study.

BACKGROUND: The growing amount of high dimensional biomolecular data has spawned new statistical and computational models for risk prediction and disease classification. Yet, many of these methods do not yield biologically interpretable models, despite offering high classification accuracy. An exception, the top-scoring pair (TSP) algorithm derives parameter-free, biologically interpretable single pair decision rules that are accurate and robust in disease classification. However, standard TSP methods do not accommodate covariates that could heavily influence feature selection for the top-scoring pair. Herein, we propose a covariate-adjusted TSP method, which uses residuals from a regression of features on the covariates for identifying top scoring pairs. We conduct simulations and a data application to investigate our method, and compare it to existing classifiers, LASSO and random forests. RESULTS: Our simulations found that features that were highly correlated with clinical variables had high likelihood of being selected as top scoring pairs in the standard TSP setting. However, through residualization, our covariate-adjusted TSP was able to identify new top scoring pairs, that were largely uncorrelated with clinical variables. In the data application, using patients with diabetes (n = 977) selected for metabolomic profiling in the Chronic Renal Insufficiency Cohort (CRIC) study, the standard TSP algorithm identified (valine-betaine, dimethyl-arg) as the top-scoring metabolite pair for classifying diabetic kidney disease (DKD) severity, whereas the covariate-adjusted TSP method identified the pair (pipazethate, octaethylene glycol) as top-scoring. Valine-betaine and dimethyl-arg had, respectively, ≥ 0.4 absolute correlation with urine albumin and serum creatinine, known prognosticators of DKD. Thus without covariate-adjustment the top-scoring pair largely reflected known markers of disease severity, whereas covariate-adjusted TSP uncovered features liberated from confounding, and identified independent prognostic markers of DKD severity. Furthermore, TSP-based methods achieved competitive classification accuracy in DKD to LASSO and random forests, while providing more parsimonious models. CONCLUSIONS: We extended TSP-based methods to account for covariates, via a simple, easy to implement residualizing process. Our covariate-adjusted TSP method identified metabolite features, uncorrelated from clinical covariates, that discriminate DKD severity stage based on the relative ordering between two features, and thus provide insights into future studies on the order reversals in early vs advanced disease states.

High-Throughput Metabolomics and Diabetic Kidney Disease Progression: Evidence from the Chronic Renal Insufficiency (CRIC) Study.

INTRODUCTION: Metabolomics could offer novel prognostic biomarkers and elucidate mechanisms of diabetic kidney disease (DKD) progression. Via metabolomic analysis of urine samples from 995 CRIC participants with diabetes and state-of-the-art statistical modeling, we aimed to identify metabolites prognostic to DKD progression. METHODS: Urine samples (N = 995) were assayed for relative metabolite abundance by untargeted flow-injection mass spectrometry, and stringent statistical criteria were used to eliminate noisy compounds, resulting in 698 annotated metabolite ions. Utilizing the 698 metabolites' ion abundance along with clinical data (demographics, blood pressure, HbA1c, eGFR, and albuminuria), we developed univariate and multivariate models for the eGFR slope using penalized (lasso) and random forest models. Final models were tested on time-to-ESKD (end-stage kidney disease) via cross-validated C-statistics. We also conducted pathway enrichment analysis and a targeted analysis of a subset of metabolites. RESULTS: Six eGFR slope models selected 9-30 variables. In the adjusted ESKD model with highest C-statistic, valine (or betaine) and 3-(4-methyl-3-pentenyl)thiophene were associated (p < 0.05) with 44% and 65% higher hazard of ESKD per doubling of metabolite abundance, respectively. Also, 13 (of 15) prognostic amino acids, including valine and betaine, were confirmed in the targeted analysis. Enrichment analysis revealed pathways implicated in kidney and cardiometabolic disease. CONCLUSIONS: Using the diverse CRIC sample, a high-throughput untargeted assay, followed by targeted analysis, and rigorous statistical analysis to reduce false discovery, we identified several novel metabolites implicated in DKD progression. If replicated in independent cohorts, our findings could inform risk stratification and treatment strategies for patients with DKD.

Changes in plasma and urine metabolites associated with empagliflozin in patients with type 1 diabetes.

AIM: To examine the impact of the sodium-glucose co-transporter-2 inhibitor, empagliflozin, on plasma and urine metabolites in participants with type 1 diabetes. MATERIAL AND METHODS: Participants (n = 40, 50% male, mean age 24.3 years) with type 1 diabetes and without overt evidence of diabetic kidney disease had baseline assessments performed under clamped euglycaemia and hyperglycaemia, on two consecutive days. Participants then proceeded to an 8-week, open-label treatment period with empagliflozin 25 mg/day, followed by repeat assessments under clamped euglycaemia and hyperglycaemia. Plasma and urine metabolites were first grouped into metabolic pathways using MetaboAnalyst software. Principal component analysis was performed to create a representative value for each sufficiently represented metabolic group (false discovery rate ≤ 0.1) for further analysis. RESULTS: Of the plasma metabolite groups, tricarboxylic acid (TCA) cycle (P < .0001), biosynthesis of unsaturated fatty acids (P = .0045), butanoate (P < .0001), propanoate (P = .0053), and alanine, aspartate and glutamate (P < .0050) metabolites were increased after empagliflozin treatment under clamped euglycaemia. Of the urine metabolite groups, only butanoate metabolites (P = .0005) were significantly increased. Empagliflozin treatment also attenuated the increase in a number of urine metabolites observed with acute hyperglycaemia. CONCLUSIONS: Empagliflozin was associated with increased lipid and TCA cycle metabolites in participants with type 1 diabetes, suggesting a shift in metabolic substrate use and improved mitochondrial function. These effects result in more efficient energy production and may contribute to end-organ protection by alleviating local hypoxia and oxidative stress.

Multiplexed Comparative Analysis of Intact Glycopeptides Using Electron-Transfer Dissociation and Synchronous Precursor Selection Based Triple-Stage Mass Spectrometry.

A recently developed synchronous precursor selection (SPS) mass spectrometry to the third (MS3) protocol enables more accurate multiplexed quantification of proteins/peptides using tandem mass tags (TMT) through comparison of reporter ion intensities at the MS3 level. However, challenges still exist for TMT-based simultaneous quantification and identification of intact glycopeptides due to inefficient peptide backbone fragmentation when using collision-induced dissociation (CID). To overcome this limitation, here we report an improved SPS/ETD workflow for TMT-based intact glycopeptide quantification and identification. The SPS/ETD approach was implemented on an Orbitrap Tribrid mass spectrometer and begins with selection of a parent ion in the MS scan, followed by tandem mass spectrometry (MS2) fragmentation by CID in the ion trap. Following MS2 fragmentation, SPS enables simultaneous isolation of the top 10 MS2 fragment ions for further higher energy collisional dissociation (HCD) fragmentation with the resulting MS3 fragments detected in an Orbitrap analyzer. Here, in addition to the standard SPS workflow, an electron-transfer dissociation (ETD) MS2 was performed and analyzed in the ion trap. The resultant ETD and CID spectra were used for the identification of the intact glycopeptides, while the quantitative comparison of site-specific glycans was achieved utilizing TMT reporter ions from HCD MS3 spectra. For intact glycopeptides, through systematic optimization and evaluation using a glycoprotein interference model, the SPS/ETD approach was demonstrated to offer improved accuracy, precision, and sensitivity compared to traditional data-dependent MS2 quantification, while maintaining the glycopeptide identification capability. Finally, this workflow was applied for the site-specific quantitative comparison of the glycoforms for two therapeutic enzymes (Cerezyme and VPRIV) and their different lots. The results demonstrate that this workflow is suitable for TMT-based intact glycopeptide characterization of glycoproteins.

A Targeted Multiomics Approach to Identify Biomarkers Associated with Rapid eGFR Decline in Type 1 Diabetes.

BACKGROUND: Individuals with type 1 diabetes (T1D) demonstrate varied trajectories of estimated glomerular filtration rate (eGFR) decline. The molecular pathways underlying rapid eGFR decline in T1D are poorly understood, and individual-level risk of rapid eGFR decline is difficult to predict. METHODS: We designed a case-control study with multiple exposure measurements nested within 4 well-characterized T1D cohorts (FinnDiane, Steno, EDC, and CACTI) to identify biomarkers associated with rapid eGFR decline. Here, we report the rationale for and design of these studies as well as results of models testing associations of clinical characteristics with rapid eGFR decline in the study population, upon which "omics" studies will be built. Cases (n = 535) and controls (n = 895) were defined as having an annual eGFR decline of ≥3 and <1 mL/min/1.73 m2, respectively. Associations of demographic and clinical variables with rapid eGFR decline were tested using logistic regression, and prediction was evaluated using area under the curve (AUC) statistics. Targeted metabolomics, lipidomics, and proteomics are being performed using high-resolution mass-spectrometry techniques. RESULTS: At baseline, the mean age was 43 years, diabetes duration was 27 years, eGFR was 94 mL/min/1.73 m2, and 62% of participants were normoalbuminuric. Over 7.6-year median follow-up, the mean annual change in eGFR in cases and controls was -5.7 and 0.6 mL/min/1.73 m2, respectively. Younger age, longer diabetes duration, and higher baseline HbA1c, urine albumin-creatinine ratio, and eGFR were significantly associated with rapid eGFR decline. The cross-validated AUC for the predictive model incorporating these variables plus sex and mean arterial blood pressure was 0.74 (95% CI: 0.68-0.79; p < 0.001). CONCLUSION: Known risk factors provide moderate discrimination of rapid eGFR decline. Identification of blood and urine biomarkers associated with rapid eGFR decline in T1D using targeted omics strategies may provide insight into disease mechanisms and improve upon clinical predictive models using traditional risk factors.

Synthesis and detection of N-sulfonated oversulfated chondroitin sulfate in marketplace heparin.

N-sulfonated oversulfated chondroitin sulfate (NS-OSCS), recently reported as a potential threat to the heparin supply, was prepared along with its intermediate derivatives. All compounds were spiked into marketplace heparin and subjected to United States Pharmacopeia (USP) identification assays for heparin (proton nuclear magnetic resonance [(1)H NMR], chromatographic identity, % galactosamine [%GalN], anti-factor IIa potency, and anti-factor Xa/IIa ratio). The U.S. Food and Drug Administration (FDA) strong-anionic exchange high-performance liquid chromatography (SAX-HPLC) method resolved NS-OSCS from heparin and OSCS and had a limit of detection of 0.26% (w/w) NS-OSCS. The %GalN test was sensitive to the presence of NS-OSCS in heparin. Therefore, current USP heparin monograph tests (i.e., SAX-HPLC and %GalN) detect the presence of NS-OSCS in heparin.

Reduced progranulin increases tau and α-synuclein inclusions and alters mouse tauopathy phenotypes via glucocerebrosidase.

Comorbid proteinopathies are observed in many neurodegenerative disorders including Alzheimer's disease (AD), increase with age, and influence clinical outcomes, yet the mechanisms remain ill-defined. Here, we show that reduction of progranulin (PGRN), a lysosomal protein associated with TDP-43 proteinopathy, also increases tau inclusions, causes concomitant accumulation of α-synuclein and worsens mortality and disinhibited behaviors in tauopathy mice. The increased inclusions paradoxically protect against spatial memory deficit and hippocampal neurodegeneration. PGRN reduction in male tauopathy attenuates activity of ß-glucocerebrosidase (GCase), a protein previously associated with synucleinopathy, while increasing glucosylceramide (GlcCer)-positive tau inclusions. In neuronal culture, GCase inhibition enhances tau aggregation induced by AD-tau. Furthermore, purified GlcCer directly promotes tau aggregation in vitro. Neurofibrillary tangles in human tauopathies are also GlcCer-immunoreactive. Thus, in addition to TDP-43, PGRN regulates tau- and synucleinopathies via GCase and GlcCer. A lysosomal PGRN-GCase pathway may be a common therapeutic target for age-related comorbid proteinopathies.

Ketone production and excretion even during mild hyperglycemia and the impact of sodium-glucose co-transporter inhibition in type 1 diabetes.

AIMS: We aimed to determine if ketone production and excretion are increased even at mild fasting hyperglycemia in type 1 diabetes (T1D) and if these are modified by ketoacidosis risk factors, including sodium-glucose co-transporter inhibition (SGLTi) and female sex. METHODS: In secondary analysis of an 8-week single-arm open-label trial of empagliflozin (NCT01392560) we evaluated ketone concentrations during extended fasting and clamped euglycemia (4-6 mmol/L) and mild hyperglycemia (9-11 mmol/L) prior to and after treatment. Plasma and urine beta-hydroxybutyrate (BHB) concentrations and fractional excretion were analyzed by metabolomic analysis. RESULTS: Forty participants (50 % female), aged 24 ± 5 years, HbA1c 8.0 ± 0.9 % (64 ± 0.08 mmol/mol) with T1D duration of 17.5 ± 7 years, were studied. Increased BHB production even during mild hyperglycemia (median urine 6.3[3.5-13.6] vs. 3.5[2.2-7.0] µmol/mmol creatinine during euglycemia, p < 0.001) was compensated by increased fractional excretion (0.9 % [0.3-1.6] vs. 0.4 % [0.2-0.9], p < 0.001). SGLTi increased production and attenuated the increased BHB fractional excretion (decreased to 0.3 % during mild hyperglycemia, p < 0.001), resulting in higher plasma concentrations (increased to 0.21 [0.05-0.40] mmol/L, p < 0.001), particularly in females (interaction p < 0.001). CONCLUSIONS: Even mild hyperglycemia is associated with greater ketone production, compensated by urinary excretion, in T1D. However, SGLTi exaggerates production and partially reduces compensatory excretion, particularly in women.

Direct approach for qualitative and quantitative characterization of glycoproteins using tandem mass tags and an LTQ Orbitrap XL electron transfer dissociation hybrid mass spectrometer.

The application of multiplexed isobaric tandem mass tag (TMT) labeling and an LTQ Orbitrap XL ETD (electron transfer dissociation) hybrid mass spectrometer as a direct approach for qualitative and quantitative characterization of glycoproteins is reported. Bovine fetuin was used as a model glycoprotein in this study. For online liquid chromatography-mass spectrometry (LC-MS) analysis, high-resolution, mass accurate full scan MS spectra were acquired in the Orbitrap mass analyzer followed by data-dependent tandem mass spectrometry (MS/MS) with alternating collision-induced dissociation (CID), ETD, and higher-energy collisional dissociation (HCD) scans. An additional in-source dissociation scan was used as a highly sensitive and selective detection method for eluting glycosylated peptides. By alternatively using three different dissociation methods, 23 glycoforms from all 5 corresponding glycopeptides were identified from a trypsin digest of bovine fetuin. With ETD, labile glycans were retained without any signs of carbohydrate cleavage with concurrent fragmentation of the peptide backbone. Glycosylation sites were clearly localized from the ETD fragmentation data. Glycan structure elucidation was accomplished using CID. The CID experiments generated fragment ions predominantly from cleavage of glycosidic bonds without breaking the peptide bond. Novel to this method, the TMT labeling protocol was modified and adapted for higher labeling efficiency, and a TriVersa NanoMate was used to reinfuse samples to improve ETD and HCD spectra of glycopeptides. Quantification with TMT was verified based on the HCD spectra from multiple nonglycopeptides and glycopeptides. This method can be used as a qualitative and quantitative technique for direct characterization of glycoproteins and has applicability for detection of counterfeit glycoprotein drug products.

Lysosomal TMEM106B interacts with galactosylceramidase to regulate myelin lipid metabolism.

TMEM106B is an endolysosomal transmembrane protein not only associated with multiple neurological disorders including frontotemporal dementia, Alzheimer's disease, and hypomyelinating leukodystrophy but also potentially involved in COVID-19. Additionally, recent studies have identified amyloid fibrils of C-terminal TMEM106B in both aged healthy and neurodegenerative brains. However, so far little is known about physiological functions of TMEM106B in the endolysosome and how TMEM106B is involved in a wide range of human conditions at molecular levels. Here, we performed lipidomic analysis of the brain of TMEM106B-deficient mice. We found that TMEM106B deficiency significantly decreases levels of two major classes of myelin lipids, galactosylceramide and its sulfated derivative sulfatide. Subsequent co-immunoprecipitation assay showed that TMEM106B physically interacts with galactosylceramidase. We also found that galactosyceramidase activity was significantly increased in TMEM106B-deficient brains. Thus, our results reveal a novel function of TMEM106B interacting with galactosyceramidase to regulate myelin lipid metabolism and have implications for TMEM106B-associated diseases.

IL-6/JAK2-dependent G6PD phosphorylation promotes nucleotide synthesis and supports tumor growth.

OBJECTIVE: Tumor cells hijack inflammatory mechanisms to promote their own growth. IL-6 is one of the major cytokines, and is frequently upregulated in tumors. The pentose phosphate pathway (PPP) generates the indispensable building blocks to produce various nucleotides. Here we aimed to determine whether and how PPP is timely tuned in response to IL-6 to support tumor growth. METHODS: Protein expression was examined by immunoblot. Protein interaction was examined by immunoprecipitation. Tumor cell proliferation in in vitro culture was examined by BrdU assay and colony formation assay. Tumor cell proliferation in mouse xenograft model was examined by Ki-67 staining. RESULTS: Here we show that the metabolic flux of PPP and enzymatic activity of glucose-6-phosphate dehydrogenase (G6PD) is rapidly induced under IL-6 treatment, without obvious changes in G6PD expression level. Mechanistically, Janus kinase 2 (JAK2) phosphorylates G6PD Y437 under IL-6 treatment, which accentuates G6PD enzymatic activity by promoting G6PD binding with its substrate G6P. Further, JAK2-dependent G6PD Y437 phosphorylation is required for IL-6-induced nucleotide biosynthesis and tumor cell proliferation, and is associated with the progression of oral squamous cell carcinoma. CONCLUSIONS: Our findings report a new mechanism implicated in the crosstalk between tumor cells and inflammatory microenvironment, by which JAK2-dependent activation of G6PD governs nucleotide synthesis to support tumor cell proliferation, thereby highlighting its value as a potential anti-tumor target.

A technology assisted precision ketogenic diet intervention for cardio-renal-metabolic health in overweight or obese adults: Protocol for a randomized controlled trial.

BACKGROUND: The obesity epidemic is a public health concern, as it is associated with a variety of chronic conditions. The ketogenic diet has drawn much scientific and public attention. However, implementation is challenging and its effect on cardio-renal-metabolic health is inconclusive. This study will assess the feasibility, acceptability, and preliminary efficacy of a technology-assisted ketogenic diet on cardio-renal-metabolic health. METHODS: This is a single center, 6-month, stratified, randomized controlled trial. A total of 60 overweight/obese adults (18+ years old) will be enrolled, including 20 without type 2 diabetes (T2D) and without chronic kidney disease (CKD); 20 with T2D, but without CKD; and 20 with early-stage CKD. Participants will be stratified based on health conditions and randomized into a ketogenic diet (n = 30) or a low-fat diet group (n = 30). Health education involving diet and physical activity will be delivered both digitally and in-person. Mobile and connected health technologies will be used to track lifestyle behaviors and health indicators, as well as provide weekly feedback. The primary outcome (weight) and the secondary outcomes (e.g., blood pressure, glycemic control, renal health) will be assessed with traditional measurements and metabolomics. DISCUSSION: Mobile and connected health technologies provide new opportunities to improve chronic condition management, health education attendance, planned lifestyle changes and engagement, and health outcomes. The advancement of bioinformatics technology offers the possibility to profile and analyze omics data which may advance our understanding of the underlying mechanisms of intervention effects on health outcomes at the molecular level for personalized and precision lifestyle interventions.

Characterization of currently marketed heparin products: analysis of molecular weight and heparinase-I digest patterns.

We evaluated polyacrylamide gel electrophoresis (PAGE) and size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) approaches to determine weight-average molecular weight (M(w)) and polydispersity (PD) of heparins. A set of unfractionated heparin sodium (UFH) and low-molecular-weight heparin (LMWH) samples obtained from nine manufacturers which supply the US market were assessed. For SEC-MALLS, we measured values for water content, refractive index increment (dn/dc), and the second virial coefficient (A(2)) for each sample prior to molecular weight assessment. For UFH, a mean ± standard deviation value for M(w) of 16,773 ± 797 was observed with a range of 15,620 to 18,363 (n = 20, run in triplicate). For LMWHs by SEC-MALLS, we measured mean M(w) values for dalteparin, tinzaparin, and enoxaparin of 6,717 ± 71 (n = 4), 6,670 ± 417 (n = 3), and 3,959 ± 145 (n = 3), respectively. PAGE analysis of the same UFH, dalteparin, tinzaparin, and enoxaparin samples showed values of 16,135 ± 643 (n = 20), 5,845 ± 45 (n = 4), 6,049 ± 95 (n = 3), and 4,772 ± 69 (n = 3), respectively. These orthogonal measurements are the first M(w) results obtained with a large heparin sample set on product being marketed after the heparin crisis of 2008 changed the level of scrutiny of this drug class. In this study, we compare our new data set to samples analyzed over 10 years earlier. In addition, we found that the PAGE analysis of heparinase digested UFH and neat LMWH samples yield characteristic patterns that provide a facile approach for identification and assessment of drug quality and uniformity.

Characterization of currently marketed heparin products: key tests for quality assurance.

During the 2007-2008 heparin crisis, it was found that the United States Pharmacopeia (USP) testing monograph for unfractionated heparin sodium (UFH) did not detect the presence of the contaminant, oversulfated chondroitin sulfate (OSCS) in heparin. In response to this concern, new tests and specifications were developed by the Food and Drug Administration (FDA) and USP and put in place to not only detect the contaminant OSCS but also to improve assurance of quality and purity of the drug product. Additional tests were also developed to monitor the heparin supply chain for other possible economically motivated additives or impurities. In 2009, a new USP monograph was put in place that includes 500 MHz (1)H NMR, SAX-HPLC, %galactosamine in total hexosamine, and anticoagulation time assays with purified factor IIa or factor Xa. These tests represent orthogonal approaches for UFH identification, measurement of bioactivity, and for detection of process impurities or contaminants in UFH. The FDA has applied these analytical approaches to the study of UFH active pharmaceutical ingredients in the marketplace. Here, we describe results from a comprehensive survey of UFH collected from seven different sources after the 2009 monograph revision and compare these data with results obtained on other heparin samples collected during the 2007-2008 crisis.

Overview of Lipidomic Analysis of Triglyceride Molecular Species in Biological Lipid Extracts.

Triglyceride (TG) is a class of neutral lipids, which functions as an energy storage depot and is important for cellular growth, metabolism, and function. The composition and content of TG molecular species are crucial factors for nutritional aspects in food chemistry and are directly associated with several diseases, including atherosclerosis, diabetes, obesity, stroke, etc. As a result of the complexities of aliphatic moieties and their different connections/locations to the glycerol backbone in TG molecules, accurate identification of individual TG molecular species and quantitative assessment of TG composition and content are particularly challenging, even at the current stage of lipidomics development. Herein, methods developed for analysis of TG species, such as liquid chromatography-mass spectrometry with a variety of columns and different mass spectrometric techniques, shotgun lipidomics approaches, and ion-mobility-based analysis, are reviewed. Moreover, the potential limitations of the methods are discussed. It is our sincere hope that the overviews and discussions can provide some insights for researchers to select an appropriate approach for TG analysis and can serve as the basis for those who would like to establish a methodology for TG analysis or develop a new method when novel tools become available. Biologically accurate analysis of TG species with an enabling method should lead us toward improving the nutritional quality, revealing the effects of TG on diseases, and uncovering the underlying biochemical mechanisms related to these diseases.

Qualitative and quantitative comparison of brand name and generic protein pharmaceuticals using isotope tags for relative and absolute quantification and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry.

The capability of iTRAQ (isotope tags for relative and absolute quantification) reagents coupled with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) as a qualitative and quantitative technique for the analysis of complicated protein pharmaceutical mixtures was evaluated. Mixtures of Somavert and Miacalcin with a small amount of bovine serum albumin (BSA) as an impurity were analyzed. Both Somavert and Miacalcin were qualitatively identified, and BSA was detected at levels as low as 0.8mol%. Genotropin and Somavert were compared in a single experiment, and all of the distinct amino acid residues from the two proteins were readily identified. Four somatropin drug products (Genotropin, Norditropin, Jintropin, and Omnitrope) were compared using the iTRAQ/MALDI-MS method to determine the similarity between their primary structures and quantify the amount of protein in each product. All four product samples were well labeled and successfully compared when a filtration cleanup step preceded iTRAQ labeling. The quantitative accuracy of the iTRAQ method was evaluated. In all cases, the accuracy of experimentally determined protein ratios was higher than 90%, and the relative standard deviation (RSD) was less than 10%. The iTRAQ and global internal standard technology (GIST) methods were compared, and the iTRAQ method provided both higher sequence coverage and enhanced signal intensity.

The impact of standard accelerated stability conditions on antibody higher order structure as assessed by mass spectrometry.

Protein therapeutic higher order structure (HOS) is a quality attribute that can be assessed to help predict shelf life. To model product shelf-life values, possible sample-dependent pathways of degradation that may affect drug efficacy or safety need to be evaluated. As changes in drug thermal stability over time can be correlated with an increased risk of HOS perturbations, the effect of long-term storage on the product should be measured as a function of temperature. Here, complementary high-resolution mass spectrometry methods for HOS analysis were used to identify storage-dependent changes of biotherapeutics (bevacizumab (Avastin), trastuzumab (Herceptin), rituximab (Rituxan), and the NIST reference material 8671 (NISTmAb)) under accelerated or manufacturer-recommended storage conditions. Collision-induced unfolding ion mobility-mass spectrometry data showed changes in monoclonal antibody folded stability profiles that were consistent with the appearance of a characteristic unfolded population. Orthogonal hydrogen-deuterium exchange-mass spectrometry data revealed that the observed changes in unfolding occurred in parallel to changes in HOS localized to the periphery of the hinge region. Using intact reverse-phase liquid chromatography-mass spectrometry, we identified several mass species indicative of peptide backbone hydrolysis, located between the variable and constant domains of the heavy chain of bevacizumab. Taken together, our data highlighted the capability of these approaches to identify age- or temperature-dependent changes in biotherapeutic HOS.

Detection of protein modifications and counterfeit protein pharmaceuticals using isotope tags for relative and absolute quantification and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry: studies of insulins.

Isotope tags for relative and absolute quantification (iTRAQ) reagent coupled with matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometric analysis has been evaluated as both a qualitative and quantitative method for the detection of modifications to active pharmaceutical ingredients derived from recombinant DNA technologies and as a method to detect counterfeit drug products. Five types of insulin (human, bovine, porcine, Lispro, and Lantus) were used as model products in the study because of their minor variations in amino acid sequence. Several experiments were conducted in which each insulin variant was separately digested with Glu-C, and the digestate was labeled with one of four different iTRAQ reagents. All digestates were then combined for desalting and MALDI-TOF/TOF mass spectrometric analysis. When the digestion procedure was optimized, the insulin sequence coverage was 100%. Five different types of insulin were readily differentiated, including human insulin (P28K29) and Lispro insulin (K28P29), which differ only by the interchange of two contiguous residues. Moreover, quantitative analyses show that the results obtained from the iTRAQ method agree well with those determined by other conventional methods. Collectively, the iTRAQ method can be used as a qualitative and quantitative technique for the detection of protein modification and counterfeiting.

Treatment of 60 cases of gouty arthritis with modified Simiao Tang.

OBJECTIVE: To observe the clinical effect of a modified Simiao Tang (Modified Decoction of Four Wonderful Drugs) for gouty arthritis and its influence on uric acid in blood. METHODS: 120 cases of gouty arthritis were randomly divided into the treatment group and control group with 60 cases in each group. Modified Simiao Tang (MST) was orally administered to the patients in the treatment group and allopurinol tablet was orally administered to the patients in the control group. The clinical effects of two groups were evaluated after one-week treatment and uric acid (UA) and C-reactive protein (CRP) levels in blood were determined after 1-month treatment. RESULTS: The total effective rate in the treatment group was significantly higher than in the control group, 86.7% vs. 68.3% (P < 0.01). And the treatment group was also significantly better than the control group in decreasing UA and CRP (P < 0.05 or P < 0.01). CONCLUSIONS: MST can significantly improve the symptoms and signs of gouty arthritis and decrease the levels of UA and CRP. It is good for gouty arthritis.

A General LC-MS/MS Method for Monitoring Potential ß-Lactam Contamination in Drugs and Drug-Manufacturing Surfaces.

Penicillins and some non-penicillin ß-lactams may cause potentially life-threatening allergic reactions. Thus, possible cross contamination of ß-lactams in food or drugs can put people at risk. Therefore, when there is a reasonable possibility that a non-penicillin product could be contaminated by penicillin, the drug products are tested for penicillin contamination. Here, a sensitive and rapid method for simultaneous determination of multiple ß-lactam antibiotics using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated. Mass spectral acquisition was performed on a Q-Exactive HF mass spectrometer in positive ion mode with parallel reaction monitoring (PRM). The method was validated for seven ß-lactam antibiotics including one or two from each class and a synthetic intermediate. The quantification precision and accuracy at 200 ppb were in the range of ± 1.84 to ± 4.56 and - 5.20 to 3.44%, respectively. The limit of detection (LOD) was 0.2 ppb, and the limit of quantitation (LOQ) was 2 ppb with a linear dynamic range (LDR) of 2-2000 ppb for all eight ß-lactams. From various drug products, the recoveries of eight ß-lactams at 200 and 2 ppb ranged from 93.8 ± 3.2 to 112.1 ± 4.2% and 89.7 ± 4.6 to 110.6 ± 1.9%, respectively. The application of the method for detecting cross contamination of trace ß-lactams (0.2 ppb) and for monitoring facility surface cleaning was also investigated. This sensitive and fast method was fit-for-purpose for detecting and quantifying trace amount of ß-lactam contamination, monitoring cross contamination in manufacturing processes, and determining potency for regulatory purposes and for quality control.