Delta-S-Cys-Albumin as a Marker of Pediatric Biospecimen Integrity.

Blood plasma storage is a crucial element of pediatric biobanking. Improperly stored or handled specimens (e.g., at > -30°C) can result in altered biomolecular compositions that no longer reflects in vivo reality. We report application of a previously developed assay in adults-the ΔS-Cys-Albumin assay, which facilitates estimation of plasma and serum exposure to thawed conditions-to a population of pediatric EDTA plasma samples from patients aged 3-18 years to determine the assay's applicability, estimate its reference range for pediatric samples, and assess the impact of pre-centrifugation delay at 0°C. In addition, the effect of plasma thawed-state exposure to a range of times at 23°C, 4°C, and -20°C on ΔS-Cys-Albumin was evaluated. Using 98 precollected and processed pediatric EDTA plasma specimens, no difference was found in ΔS-Cys-Albumin under conditions of pre-centrifugation delay for up to 10 hours at 0°C. This lack of change allowed us to estimate a pediatric reference range for ΔS-Cys-Albumin of 7.0%-22.5% (mean of 12.8%) with a modest Pearson correlation between ΔS-Cys-Albumin and age (p = 0.0037, R2 = 0.29). ΔS-Cys-Albumin stability in six specimens at 23°C, 4°C, and -20°C was also evaluated. Plateaus in the decay curves were reached by 1 day, 7 days, and 14-28 days at these respective temperatures. The estimated pediatric reference range observed in children was lower than that previously observed in 180 adults of 12.3%-30.6% (mean of 20.0%), and the slope of the age correlation in children was twice as steep as that from adults. ΔS-Cys-Albumin decay curves at 23°C, 4°C, and -20°C were similar to those previously observed in adults. The data reported here support the use of ΔS-Cys-Albumin in evaluating the integrity and overall exposure of pediatric EDTA plasma specimens to thawed conditions. In doing so, they add an important quality control tool to the biobanker's arsenal.

Glycan node profiling of soluble and membrane glycoproteins in whole cell lysates.

Glycan node analysis (GNA) is a molecularly bottom-up glycomics technique based on the relative quantification of glycan linkage-specific monosaccharide units ("glycan nodes"). It was originally applied to blood plasma/serum, where it detected and predicted progression, reoccurrence, and survival in different types of cancer. Here, we have adapted this technology to previously inaccessible membrane glycoproteins from cultured cells. The approach is facilitated by methanol/chloroform precipitation of cell lysates and a "liquid phase permethylation" (LPP) procedure. LPP gave better signal-to-noise, yield and precision for most of the glycan nodes from membrane glycoproteins/glycolipids than the conventional solid phase permethylation approach. This GNA approach in cell lysates revealed that specific glycan features such as antennary fucosylation, N-glycan branching, and α2,6-sialylation were elevated in hepatocellular carcinoma (HepG2) cells relative to leukemia cells (THP-1 and K562) and normal donor PBMCs. Additional nodes commonly associated with glycolipids were elevated in the leukemia cells relative to HepG2 cells and PBMCs. Exposure of HepG2 cells to a fucosyltransferase inhibitor resulted in a significant reduction in the relative abundance of 3,4-substituted GlcNAc, which represents antennary fucosylation-providing further proof-of-concept that downregulation of glycosyltransferase activity is detected by shifts in glycan node expression-now detectable in membrane glycoproteins.

Glycan Node Analysis Detects Varying Glycosaminoglycan Levels in Melanoma-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) play important roles in (patho)physiological processes by mediating cell communication. Although EVs contain glycans and glycosaminoglycans (GAGs), these biomolecules have been overlooked due to technical challenges in comprehensive glycome analysis coupled with EV isolation. Conventional mass spectrometry (MS)-based methods are restricted to the assessment of N-linked glycans. Therefore, methods to comprehensively analyze all glyco-polymer classes on EVs are urgently needed. In this study, tangential flow filtration-based EV isolation was coupled with glycan node analysis (GNA) as an innovative and robust approach to characterize most major glyco-polymer features of EVs. GNA is a molecularly bottom-up gas chromatography-MS technique that provides unique information that is unobtainable with conventional methods. The results indicate that GNA can identify EV-associated glyco-polymers that would remain undetected with conventional MS methods. Specifically, predictions based on GNA identified a GAG (hyaluronan) with varying abundance on EVs from two different melanoma cell lines. Enzyme-linked immunosorbent assays and enzymatic stripping protocols confirmed the differential abundance of EV-associated hyaluronan. These results lay the framework to explore GNA as a tool to assess major glycan classes on EVs, unveiling the EV glycocode and its biological functions.

Oxidized LDL is stable in human serum under extended thawed-state conditions ranging from -20 °C to room temperature.

Introduction: Oxidized LDL (oxLDL) is formed by the spontaneous reaction between aldehyde byproducts of lipid peroxidation and lysine residues of apolipoprotein B within LDL. Clinically, oxLDL is used as a marker of coronary artery disease and predictor of metabolic syndrome risk. Despite its popularity as a clinical marker, no systematic studies of oxLDL stability, in which serum or plasma has been pre-analytically exposed to an array of different time and temperature conditions, have been carried out. Objective: To systematically evaluate the stability of oxLDL in human serum samples exposed to thawed conditions (> -30 °C) for varying periods of time while monitoring a second protein/small molecule redox system as a positive control for non-enzymatic biomolecular activity. Methods: OxLDL was measured in serum samples, from 24 different humans, that had been pre-exposed to three different time courses at 23 °C, 4 °C and -20 °C using ELISA kits from Mercodia that employ the 4E6 mouse monoclonal antibody. A liquid chromatography/mass spectrometry-based marker of serum exposure to thawed conditions known as ΔS-Cys-Albumin was employed as a positive control. Results: OxLDL was stable in serum exposed to 23 °C for up to 48 h, 4 °C for 21 days, or -20 °C for 65 days. ΔS-Cys-Albumin changed dramatically during these time courses (p < 0.001). Conclusions: OxLDL is remarkably stable ex vivo in human serum samples exposed to thawed conditions.

Tracking the Stability of Clinically Relevant Blood Plasma Proteins with Delta-S-Cys-Albumin-A Dilute-and-Shoot LC/MS-Based Marker of Specimen Exposure to Thawed Conditions.

Biomolecular integrity can be compromised when blood plasma/serum (P/S) specimens are improperly handled. Compromised analytes can subsequently produce erroneous results-without any indication of having done so. We recently introduced an LC/MS-based marker of P/S exposure to thawed conditions called ΔS-Cys-Albumin which, aided by an established rate law, quantitatively tracks exposure of P/S to temperatures greater than their freezing point of -30 °C. The purposes of this study were to (1) evaluate ΔS-Cys-Albumin baseline values in gastrointestinal cancer patients and cancer-free control donors, (2) empirically assess the kinetic profiles of ΔS-Cys-Albumin at 23 °C, 4 °C, and -20 °C, and (3) empirically link ΔS-Cys-Albumin to the stability of clinically relevant proteins. ΔS-Cys-Albumin was measured at ≥ 9 different time points per exposure temperature in serum and K2EDTA plasma samples from 24 separate donors in aliquots kept separately at 23 °C, 4 °C, and -20 °C. Twenty-one clinically relevant plasma proteins were measured at four time points per temperature via a multiplexed immunoassay on the Luminex platform. Protein stability was assessed by mixed effects models. Coordinated shifts in stability between ΔS-Cys-Albumin and the unstable proteins were documented by repeated measures and Pearson correlations. Plasma ΔS-Cys-Albumin dropped from approximately 20% to under 5% within 96 h at 23 °C, 28 days at 4 °C, and 65 days at -20 °C. On average, 22% of the 21 proteins significantly changed in apparent concentration at each exposure temperature (p < 0.0008 with >10% shift). A linear inverse relationship was found between the percentage of proteins destabilized and ΔS-Cys-Albumin (r = -0.61; p < 0.0001)-regardless of the specific time/temperature of exposure. ΔS-Cys-Albumin tracks cumulative thawed-state exposure. These results now enable ΔS-Cys-Albumin to approximate the percentage of clinically relevant proteins that have been compromised by incidental plasma exposure to thawed-state conditions.

Extracellular vesicle glucose transporter-1 and glycan features in monocyte-endothelial inflammatory interactions.

Monocyte-induced endothelial cell inflammation is associated with multiple pathological conditions, and extracellular vesicles (EVs) are essential nanosized components of intercellular communication. EVs derived from endotoxin-stimulated monocytes were previously shown to carry pro-inflammatory proteins and RNAs. The role of glucose transporter-1 (GLUT-1) and glycan features in monocyte-derived EV-induced endothelial cell inflammation remains largely unexplored. This study demonstrates that EVs derived from endotoxin-stimulated monocytes activate inflammatory pathways in endothelial cells, which are partially attributed to GLUT-1. Alterations in glycan features and increased levels of GLUT-1 were observed in EVs derived from endotoxin-stimulated monocytes. Notably, inhibition of EV-associated GLUT-1, through the use of fasentin, suppressed EV-induced inflammatory cytokines in recipient endothelial cells.

Oxidized-Desialylated Low-Density Lipoprotein Inhibits the Antitumor Functions of Lymphokine Activated Killer Cells.

Elevated concentrations of circulating low density lipoprotein (LDL) that is abnormally oxidized and desialylated is both a precursor to and a hallmark of atherosclerosis. Peripheral blood mononuclear cells (PBMCs) treated in vitro with interleukin-2 (IL-2) become lymphokine activated killer (LAK) cells, the primary effectors of which are NK cells and NKT cells. LAK cells display antitumor functions such as increased cytotoxicity and IFN-γ production, and they have been evaluated as a potential cancer therapeutic. Atherosclerotic processes may influence innate immunity against cancer. Because prior studies have shown that low density lipoprotein (LDL) reduces T-cell and NK cell antitumor functions, we asked whether oxidized-desialylated LDL affects the functionality of LAK cells in vitro. We show here that LAK cells take up oxidized-desialylated LDL to a significantly greater extent than native LDL over a period of 72 hours. This resulted in a significant downregulation of LAK cell cytotoxicity against K562 cells. In particular, the expression of IFN-γ, CD56, and NKG2D were reduced upon oxidized-desialylated LDL treatment of LAK cells and, conversely, their expression was enhanced with native LDL. It was also observed that as the number of CD56 and NKG2D positive cells decreased upon treatment with oxidized-desialylated LDL, the number of CD3 positive cells increased in proportion. Additionally, only a slight inhibition of LAK cell cytotoxicity was observed with desialylation alone of LDL, and no significant inhibition was observed with oxidation alone of LDL. Thus, this study describes a new role of oxidized-desialylated LDL as an inhibitor of the antitumor functions of LAK cells. These observations have implications for how atherosclerosis processes, namely oxidation and desialylation of LDL, may influence LAK cell antitumor activity.

Glycosylation Profiling of Glycoproteins Secreted from Cultured Cells Using Glycan Node Analysis and GC-MS.

Glycan "node" analysis is the process by which pooled glycans within complex biological samples are chemically deconstructed in a way that facilitates the analytical quantification of uniquely linked monosaccharide units (glycan "nodes"). It is based on glycan methylation analysis (a.k.a. linkage analysis) that has historically been applied to pre-isolated glycans. Thus, when using glycan node analysis, unique glycan features within whole biospecimens such as "core fucosylation," "α2-6 sialylation," "ß1-6 branching," "ß1-4 branching," and "bisecting GlcNAc," are captured as single analytical signals by GC-MS. Here we describe the use of this methodology in cell culture supernatant and in the analysis of IgG (alpha-1 antitrypsin) glycans. The effect of IL-6 and IL-1ß cytokines on secreted hepatocyte protein glycan features is demonstrated; likewise, the impact of neuraminidase treatment of IgG is illustrated. For the majority of glycan nodes, the assay is consistent and reproducible on a day-to-day basis; because of this, relatively subtle shifts in the relative abundance of glycan features can be captured using this approach.

Relationship Between Chronic Kidney Disease, Glucose Homeostasis, and Plasma Osteocalcin Carboxylation and Fragmentation.

OBJECTIVE: Chronic kidney disease (CKD) is associated with reduced insulin sensitivity, through mechanisms that are not well understood. Low vitamin K intake and incomplete carboxylation of the vitamin K-dependent protein osteocalcin may promote insulin resistance. We assessed relationships of osteocalcin concentration, carboxylation, and fragmentation with CKD and glucose homeostasis in a cross-sectional study. METHODS: We included 87 participants without diabetes: 50 (27 female) with moderate to severe CKD (estimated glomerular filtration rate <60 mL/min/1.73 m2 not treated with dialysis) and 37 (17 female) healthy controls. Total osteocalcin was measured by immunoassay, and osteocalcin carboxylation and fragmentation status by liquid chromatography-electrospray ionization-based mass spectrometric immunoassay. Endpoints included glucose tolerance (based on 2-hour oral glucose tolerance test), insulin sensitivity (hyperinsulinemic-euglycemic clamp), and pancreatic beta-cell function (intravenous glucose tolerance test). RESULTS: The total plasma osteocalcin concentration was higher in the CKD group (mean [standard deviation] 102.9 [147.5]) than that in the control group (53.6 [51.1] ng/mL, P = .03), and more osteocalcin was circulating as fragments. The extent of osteocalcin carbocylation did not differ between individuals with and without CKD. Osteocalcin concentration, carboxylation, and fragmentation were not associated with any measure of glucose homeostasis in multivariable-adjusted analyses. CONCLUSIONS: In CKD, circulating osteocalcin concentrations are elevated, in part due to larger proportions of fragmented forms. However, osteocalcin carboxylation status is not significantly different between individuals with and without CKD. Our data also do not provide support for the hypothesis that differences in osteocalcin carboxylation may explain reduced insulin sensitivity in individuals with CKD.

Glycan Node Analysis of Plasma-Derived Extracellular Vesicles.

Blood plasma is a readily accessible source of extracellular vesicles (EVs), i.e., cell-secreted nanosized carriers that contain various biomolecules, including glycans. Previous studies have demonstrated that glycans play a major role in physiological and pathological processes, and certain plasma glycans have been associated with disease conditions. However, glycome studies have been limited by a lack of analytical techniques with the throughput capacity necessary to study hundreds of clinical samples. This study is the first to characterize the EV plasma glycome based on all major glycan classes. The results based on glycan node analysis revealed, as expected, that plasma-derived EVs have distinct glycan features from donor-matched whole plasma. Specifically, glycan nodes corresponding to those observed in chondroitin sulfate, dermatan sulfate, type I keratan sulfate, and type II keratan sulfate were enriched on EVs. The identification of specific differences in glycan features in plasma vs. plasma-derived EVs is relevant for understanding the physiological role of EVs and as a reference for future diagnostic studies. Additionally, the results indicate that EV glycan nodes do not substantially differ among a small set of healthy donors. These results lay the framework for the further evaluation of all EV glycan classes as diagnostic markers, therapeutic targets, and biologically active components in health and disease.

Structural Insights into How Protein Environments Tune the Spectroscopic Properties of a Noncanonical Amino Acid Fluorophore.

Genetically encoded fluorescent noncanonical amino acids (fNCAAs) could be used to develop novel fluorescent sensors of protein function. Previous efforts toward this goal have been limited by the lack of extensive physicochemical and structural characterizations of protein-based sensors containing fNCAAs. Here, we report the steady-state spectroscopic properties and first structural analyses of an fNCAA-containing Fab fragment of the 5c8 antibody, which binds human CD40L. A previously reported 5c8 variant in which the light chain residue IleL98 is replaced with the fNCAA l-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) exhibits a 1.7-fold increase in fluorescence upon antigen binding. Determination and comparison of the apparent pKas of 7-HCAA in the unbound and bound forms indicate that the observed increase in fluorescence is not the result of perturbations in pKa. Crystal structures of the fNCAA-containing Fab in the apo and bound forms reveal interactions between the 7-HCAA side chain and surrounding residues that are disrupted upon antigen binding. This structural characterization not only provides insight into the manner in which protein environments can modulate the fluorescence properties of 7-HCAA but also could serve as a starting point for the rational design of new fluorescent protein-based reporters of protein function.

Biochemically Tracked Variability of Blood Plasma Thawed-State Exposure Times in a Multisite Collection Study.

The integrity of blood plasma/serum (P/S) specimens can be impacted by preanalytical handling and storage conditions that result in thawed-state exposures (> -30°C). We recently reported a simple dilute-and-shoot, intact-protein liquid chromatography/mass spectrometry (LC/MS) assay called ΔS-Cys-Albumin that quantifies cumulative exposure of P/S to thawed conditions based on the change in relative abundance of the oxidized (S-cysteinylated) proteoform of albumin (S-Cys-Albumin) in the native sample to that of an aliquot of the sample intentionally driven to its maximum oxidation state. Herein, we evaluated the effect of prestorage delay and initial storage temperature on sample integrity by applying the ΔS-Cys-Albumin assay to a set of plasma samples (n = 413) collected under a single clinical study but from 12 different collection sites. Major differences (p < 0.0001) were observed between different groups of samples with modestly inconsistent initial handling conditions (i.e., initial processing of whole blood to plasma and placement at -80°C completed in under 3 hours, 3-13 hours, and over 17 hours). ΔS-Cys-Albumin was significantly inversely correlated with delay time at 4°C before centrifugation and total delay before final storage at -80°C (p < 0.0001). Samples from two collection sites had much lower ΔS-Cys-Albumin values relative to samples from other sites, in accordance with the fact that they were stored at -20°C for an average of 7.6 months before shipment to the central repository for final storage at -80°C. Based on the rate law for S-Cys-Albumin formation in plasma ex vivo, the average time that each plasma specimen had been exposed to the equivalent of room temperature (23°C) was back calculated from the measured ΔS-Cys-Albumin values. A survey of clinical analytes in P/S whose measured concentrations are sensitive to the initial handling/storage conditions documented in this study is provided and the ramifications of the plasma integrity findings from this multisite clinical study are discussed.

Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay.

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic metric of lipid peroxidation in biological fluids. It is often considered a good indicator of the levels of oxidative stress within a biological sample, provided that the sample has been properly handled and stored. The assay involves the reaction of lipid peroxidation products, primarily malondialdehyde (MDA), with thiobarbituric acid (TBA), which leads to the formation of MDA-TBA2 adducts called TBARS. TBARS yields a red-pink color that can be measured spectrophotometrically at 532 nm. The TBARS assay is performed under acidic conditions (pH = 4) and at 95 °C. Pure MDA is unstable, but these conditions allow the release of MDA from MDA bis(dimethyl acetal), which is used as the analytical standard in this method. The TBARS assay is a straightforward method that can be completed in about 2 h. Preparation of assay reagents are described in detail here. Budget-conscious researchers can use these reagents for multiple experiments at a low cost rather than buying an expensive TBARS assay kit that only permits construction of a single standard curve (and thus can only be used for one experiment). The applicability of this TBARS assay is shown in human serum, low density lipoproteins, and cell lysates. The assay is consistent and reproducible, and limits of detection of 1.1 µM can be reached. Recommendations for the use and interpretation of the spectrophotometric TBARS assay are provided.

Diagnostic and Prognostic Performance of Blood Plasma Glycan Features in the Women Epidemiology Lung Cancer (WELCA) Study.

Lung cancer is the leading cause of cancer death in women living in the United States, which accounts for approximately the same percentage of cancer deaths in women as breast, ovary, and uterine cancers combined. Targeted blood plasma glycomics represents a promising source of noninvasive diagnostic and prognostic biomarkers for lung cancer. Here, 208 samples from lung cancer patients and 207 age-matched controls enrolled in the Women Epidemiology Lung Cancer (WELCA) study were analyzed by a bottom-up glycan "node" analysis approach. Glycan features, quantified as single analytical signals, including 2-linked mannose, α2-6 sialylation, ß1-4 branching, ß1-6 branching, 4-linked GlcNAc, and antennary fucosylation, exhibited abilities to distinguish cases from controls (ROC AUCs: 0.68-0.92) and predict survival in patients (hazard ratios: 1.99-2.75) at all stages. Notable alterations of glycan features were observed in stages I-II. Diagnostic and prognostic glycan features were mostly independent of smoking status, age, gender, and histological subtypes of lung cancer.

Delta-S-Cys-Albumin: A Lab Test that Quantifies Cumulative Exposure of Archived Human Blood Plasma and Serum Samples to Thawed Conditions.

Exposure of blood plasma/serum (P/S) to thawed conditions (> -30 °C) can produce biomolecular changes that skew measurements of biomarkers within archived patient samples, potentially rendering them unfit for molecular analysis. Because freeze-thaw histories are often poorly documented, objective methods for assessing molecular fitness before analysis are needed. We report a 10-µl, dilute-and-shoot, intact-protein mass spectrometric assay of albumin proteoforms called "ΔS-Cys-Albumin" that quantifies cumulative exposure of archived P/S samples to thawed conditions. The relative abundance of S-cysteinylated (oxidized) albumin in P/S increases inexorably but to a maximum value under 100% when samples are exposed to temperatures > -30 °C. The difference in the relative abundance of S-cysteinylated albumin (S-Cys-Alb) before and after an intentional incubation period that drives this proteoform to its maximum level is denoted as ΔS-Cys-Albumin. ΔS-Cys-Albumin in fully expired samples is zero. The range (mean ± 95% CI) observed for ΔS-Cys-Albumin in fresh cardiac patient P/S (n = 97) was, for plasma 12-29% (20.9 ± 0.75%) and for serum 10-24% (15.5 ± 0.64%). The multireaction rate law that governs S-Cys-Alb formation in P/S was determined and shown to predict the rate of formation of S-Cys-Alb in plasma and serum samples-a step that enables back-calculation of the time at which unknown P/S specimens have been exposed to room temperature. A blind challenge demonstrated that ΔS-Cys-Albumin can detect exposure of groups (n = 6 each) of P/S samples to 23 °C for 2 h, 4 °C for 16 h, or -20 °C for 24 h-and exposure of individual specimens for modestly increased times. An unplanned case study of nominally pristine serum samples collected under NIH-sponsorship demonstrated that empirical evidence is required to ensure accurate knowledge of archived P/S biospecimen storage history.

Behavior of blood plasma glycan features in bladder cancer.

Despite systemic therapy and cystectomy, bladder cancer is characterized by a high recurrence rate. Serum glycomics represents a promising source of prognostic markers for monitoring patients. Our approach, which we refer to as "glycan node analysis", constitutes the first example of molecularly "bottom-up" glycomics. It is based on a global glycan methylation analysis procedure that is applied to whole blood plasma/serum. The approach detects and quantifies partially methylated alditol acetates arising from unique glycan features such as α2-6 sialylation, ß1-4 branching, and core fucosylation that have been pooled together from across all intact glycans within a sample into a single GC-MS chromatographic peak. We applied this method to 122 plasma samples from former and current bladder cancer patients (n = 72 former cancer patients with currently no evidence of disease (NED); n = 38 non-muscle invasive bladder cancer (NMIBC) patients; and n = 12 muscle invasive bladder cancer (MIBC) patients) along with plasma from 30 certifiably healthy living kidney donors. Markers for α2-6 sialylation, ß1-4 branching, ß1-6 branching, and outer-arm fucosylation were able to separate current and former (NED) cases from certifiably healthy controls (ROC curve c-statistics ~ 0.80); but NED, NMIBC, and MIBC were not distinguished from one another. Based on the unexpectedly high levels of these glycan nodes in the NED patients, we hypothesized that recurrence of this disease could be predicted by some of the elevated glycan features. Indeed, α2-6 sialylation and ß1-6 branching were able to predict recurrence from the NED state using a Cox proportional hazards regression model adjusted for age, gender, and time from cancer. The levels of these two glycan features were correlated to C-reactive protein concentration, an inflammation marker and known prognostic indicator for bladder cancer, further strengthening the link between inflammation and abnormal plasma protein glycosylation.

Stage Dependence, Cell-Origin Independence, and Prognostic Capacity of Serum Glycan Fucosylation, ß1-4 Branching, ß1-6 Branching, and α2-6 Sialylation in Cancer.

Glycans represent a promising but only marginally accessed source of cancer markers. We previously reported the development of a molecularly bottom-up approach to plasma and serum (P/S) glycomics based on glycan linkage analysis that captures features such as α2-6 sialylation, ß1-6 branching, and core fucosylation as single analytical signals. Based on the behavior of P/S glycans established to date, we hypothesized that the alteration of P/S glycans observed in cancer would be independent of the tissue in which the tumor originated yet exhibit stage dependence that varied little between cancers classified on the basis of tumor origin. Herein, the diagnostic utility of this bottom-up approach as applied to lung cancer patients (n = 127 stage I; n = 20 stage II; n = 81 stage III; and n = 90 stage IV) as well as prostate (n = 40 stage II), serous ovarian (n = 59 stage III), and pancreatic cancer patients (n = 15 rapid autopsy) compared to certifiably healthy individuals (n = 30), nominally healthy individuals (n = 166), and risk-matched controls (n = 300) is reported. Diagnostic performance in lung cancer was stage-dependent, with markers for terminal (total) fucosylation, α2-6 sialylation, ß1-4 branching, ß1-6 branching, and outer-arm fucosylation most able to differentiate cases from controls. These markers behaved in a similar stage-dependent manner in other types of cancer as well. Notable differences between certifiably healthy individuals and case-matched controls were observed. These markers were not significantly elevated in liver fibrosis. Using a Cox proportional hazards regression model, the marker for α2-6 sialylation was found to predict both progression and survival in lung cancer patients after adjusting for age, gender, smoking status, and stage. The potential mechanistic role of aberrant P/S glycans in cancer progression is discussed.

Ex vivo instability of glycated albumin: A role for autoxidative glycation.

Ex vivo protein modifications occur within plasma and serum (P/S) samples due to prolonged exposure to the thawed state-which includes temperatures above -30 °C. Herein, the ex vivo glycation of human serum albumin from healthy and diabetic subjects was monitored in P/S samples stored for hours to months at -80 °C, -20 °C, and room temperature, as well as in samples subjected to multiple freeze-thaw cycles, incubated at different surface area-to-volume ratios or under different atmospheric compositions. A simple dilute-and-shoot method utilizing trap-and-elute LC-ESI-MS was employed to determine the relative abundances of the glycated forms of albumin-including forms of albumin bearing more than one glucose molecule. Significant increases in glycated albumin were found to occur within hours at room temperature, and within days at -20 °C. These increases continued over a period of 1-2 weeks at room temperature and over 200 days at -20 °C, ultimately resulting in a doubling of glycated albumin in both healthy and diabetic patients. It was also shown that samples stored at lower surface area-to-volume ratios or incubated under a nitrogen atmosphere experienced less rapid glucose adduction of albumin-suggesting a role for oxidative glycation in the ex vivo glycation of albumin.

A spin column-free approach to sodium hydroxide-based glycan permethylation.

Glycan permethylation was introduced as a tool to facilitate the study of glycans in 1903. Since that time, permethylation procedures have been continually modified to improve permethylation efficiency and qualitative applicability. Typically, however, either laborious preparation steps or cumbersome and uneconomical spin columns have been needed to obtain decent permethylation yields on small glycan samples. Here we describe a spin column-free (SCF) glycan permethylation procedure that is applicable to both O- and N-linked glycans and can be employed upstream to intact glycan analysis by MALDI-MS, ESI-MS, or glycan linkage analysis by GC-MS. The SCF procedure involves neutralization of NaOH beads by acidified phosphate buffer, which eliminates the risk of glycan oxidative degradation and avoids the use of spin columns. Optimization of the new permethylation procedure provided high permethylation efficiency for both hexose (>98%) and HexNAc (>99%) residues-yields which were comparable to (or better than) those of some widely-used spin column-based procedures. A light vs. heavy labelling approach was employed to compare intact glycan yields from a popular spin-column based approach to the SCF approach. Recovery of intact N-glycans was significantly better with the SCF procedure (p < 0.05), but overall yield of O-glycans was similar or slightly diminished (p < 0.05 for tetrasaccharides or smaller). When the SCF procedure was employed upstream to hydrolysis, reduction and acetylation for glycan linkage analysis of pooled glycans from unfractionated blood plasma, analytical reproducibility was on par with that from previous spin column-based "glycan node" analysis results. When applied to blood plasma samples from stage III-IV breast cancer patients (n = 20) and age-matched controls (n = 20), the SCF procedure facilitated identification of three glycan nodes with significantly different distributions between the cases and controls (ROC c-statistics > 0.75; p < 0.01). In summary, the SCF permethylation procedure expedites and economizes both intact glycan analysis and linkage analysis of glycans from whole biospecimens.

Variations in native protein glycation and plasma antioxidants in several birds of prey.

Birds are an anomaly among vertebrates as they are remarkably long-lived despite having naturally high blood glucose and metabolic rates. For mammals, hyperglycemia leads to oxidative stress and protein glycation. In contrast, many studies have shown that domestic and wild birds are relatively resistant to these glucose-mediated pathologies. Surprisingly very little research has examined protein glycation in birds of prey, which by nature consume a diet high in protein and fat that promotes gluconeogenesis. The purpose of this study was to evaluate protein glycation and antioxidant concentrations in serum samples from several birds of prey (bald eagle (BAEA), red-tailed hawk (RTHA), barred owl (BAOW), great horned owl (GHOW)) as protein glycation can accelerate oxidative stress and vice versa. Serum glucose was measured using a commercially available assay, native albumin glycation was measured by mass spectrometry and various antioxidants (uric acid, vitamin E, retinol and several carotenoids) were measured by high performance liquid chromatography. Although glucose concentrations were not significantly different between species (p=0.340), albumin glycation was significantly higher (p=0.004) in BAEA (23.67±1.90%) and BAOW (24.28±1.43%) compared to RTHA (14.31±0.63%). Of the antioxidants examined, lutein was significantly higher in BAOW (p=0.008). BAEA had the highest beta-cryptoxanthin and beta-carotene concentrations (p<0.005). The high concentrations of antioxidants in these birds of prey relative to other birds likely helps protect from complications that may otherwise arise from having high glucose and protein glycation.