Characterization of Glycation Adducts On Human Serum Albumin by Matrix Assisted Laser Desorption/ Ionization Time-Of-Flight Mass Spectrometry

Background—Non-enzymatic glycation of human serum albumin (HSA) is associated with the long-term complications of diabetes. We examined the structure and location of modifications on minimally glycated HSA and considered their possible impact on the binding of drugs to this protein. Methods—Minimally glycated and normal HSA (used as a control) were digested with trypsin, Glu-C or Lys-C, followed by fractionation of the resulting peptides and their analysis by matrixassisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) to determine the structures and locations of glycation adducts. Results—Several specific lysine and arginine residues were identified as modification sites in minimally glycated HSA. Residues K12, K51, K199, K205, K439 and K538 were found to be modified through the formation of fructosyl-lysine, while the modification of K159 and K286 involved the formation of pyrraline, Nε-carboxymethyl-lysine respectively. Lysine K378 was found to give Nε-carboxyethyl-lysine in some forms of glycated HSA but fructosyl-lysine in other forms. Residues R160 and R472 produced a modification based on Nε-(5-hydro-4-imidazolon-2-yl) ornithine. Lysine R222 was modified to produce argpyrimidine, Nε-[5-(2,3,4-trihydroxybutyl)-5- hydro-4-imidazolon-2-yl]ornithine or tetrahydropyrimidine. Conclusions—With the exception of K12, K199, K378, K439 and K525, all of the observed sites of modification for minimally glycated HSA were new to this current study. The fact that many of these glycation-related modifications are located at or near known drug binding sites on HSA explains why some differences have been previously noted in the binding of certain drugs to normal vs glycated HSA

COMPARISON OF MODIFICATION SITES FORMED ON HUMAN SERUM ALBUMIN AT VARIOUS STAGES OF GLYCATION

Background—Many of the complications encountered during diabetes can be linked to the nonenzymatic glycation of proteins, including human serum albumin (HSA). However, there is little information regarding how the glycation pattern of HSA changes as the total extent of glycation is varied. The goal of this study was to identify and conduct a semi-quantitative comparison of the glycation products on HSA that are produced in the presence of various levels of glycation. Methods—Three glycated HSA samples were prepared in vitro by incubating physiological concentrations of HSA with 15 mmol/l glucose for 2 or 5 weeks, or with 30 mmol/l glucose for 4 weeks. These samples were then digested and examined by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the glycation products that were formed. Results—It was found that the glycation pattern of HSA changed with its overall extent of total glycation. Many modifications including previously-reported primary glycation sites (e.g., K199, K281, and the N-terminus) were consistently found in the tested samples. Lysines 199 and 281, as well as arginine 428, contained the most consistently identified and abundant glycation products. Lysines 93, 276, 286, 414, 439, and 524/525, as well as the N-terminus and arginines 98, 197, and 521, were also found to be modified at various degrees of HSA glycation. Conclusions—The glycation pattern of HSA was found to vary with different levels of total glycation and included modifications at the 2 major drug binding sites on this protein. This result suggests that different modified forms of HSA, both in terms of the total extent of glycation and glycation pattern, may be found at various stages of diabetes. The clinical implication of these results is that the binding of HSA to some drug may be altered at various stages of diabetes as the extent of glycation and types of modifications in this protein are varied

High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time of flight mass spectrometry

Appropriate glycosylation of recombinant therapeutic glycoproteins has been emphasized in biopharmaceutical industries because the carbohydrate component can affect safety, efficacy, and consistency of the glycoproteins. Reliable quantification methods are essential to ensure consistency of their products with respect to glycosylation, particularly sialylation. Mass spectrometry (MS) has become a popular tool to analyze glycan profiles and structures, showing high resolution and sensitivity with structure identification ability. However, quantification of sialylated glycans using MS is not as reliable because of the different ionization efficiency between neutral and acidic glycans. We report here that amidation in mild acidic conditions can be used to neutralize acidic N-glycans still attached to the protein. The resulting amidated N-glycans can then released from the protein using PNGase F, and labeled with permanent charges on the reducing end to avoid any modification and the formation of metal adducts during MS analysis. The N-glycan modification, digestion, and desalting steps were performed using a single-pot method that can be done in microcentrifuge tubes or 96-well microfilter plates, enabling high throughput glycan analysis. Using this method we were able to perform quantitative MALDI-TOF MS of a recombinant human glycoprotein to determine changes in fucosylation and changes in sialylation that were in very good agreement with a normal phase HPLC oligosaccharide mapping method

High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time of flight mass spectrometry

Appropriate glycosylation of recombinant therapeutic glycoproteins has been emphasized in biopharmaceutical industries because the carbohydrate component can affect safety, efficacy, and consistency of the glycoproteins. Reliable quantification methods are essential to ensure consistency of their products with respect to glycosylation, particularly sialylation. Mass spectrometry (MS) has become a popular tool to analyze glycan profiles and structures, showing high resolution and sensitivity with structure identification ability. However, quantification of sialylated glycans using MS is not as reliable because of the different ionization efficiency between neutral and acidic glycans. We report here that amidation in mild acidic conditions can be used to neutralize acidic N-glycans still attached to the protein. The resulting amidated N-glycans can then released from the protein using PNGase F, and labeled with permanent charges on the reducing end to avoid any modification and the formation of metal adducts during MS analysis. The N-glycan modification, digestion, and desalting steps were performed using a single-pot method that can be done in microcentrifuge tubes or 96-well microfilter plates, enabling high throughput glycan analysis. Using this method we were able to perform quantitative MALDI-TOF MS of a recombinant human glycoprotein to determine changes in fucosylation and changes in sialylation that were in very good agreement with a normal phase HPLC oligosaccharide mapping method

Selection and Culture of Landscape Plants in Utah - A Guide for High Mountain Valleys

What traveler, driving across Utah, has not marveled at its diversity of geography, climate and vegetation? From Joshua-trees in the Mojave Desert, to alpine meadows, to pinion-juniper forests set against the red sandstone of the Colorado Plateau, it is truly a state of contrasts

Assessment of a spodumene ore by advanced analytical and mass spectrometry techniques to determine its amenability to processing for the extraction of lithium

A combination of analytical microscopy and mass spectrometry techniques have been used to detect and characterise different lithium minerals in a LCT-Complex spodumene-type pegmatite from Pilgangoora located in the Pilbara region of Western Australia. Information collated by these techniques can be used to predict processing amenability. Samples were categorised into three subsamples (Pil1, Pil2, Pil3) based on colour and texture having different lithologies. The mineralogy and liberation characteristics of samples were characterised using automated mineralogy techniques and the Li content and elemental distribution within minerals defined using instrumentation with secondary mass spectrometry capabilities. The majority of lithium is associated with spodumene particles with minor amounts of lithium bearing micas and beryl in the Pil1 sample, whereas in Pil2 and Pil3 spodumene is largely the lithium source. In the Pil1 sample a proportion of spodumene particles have undergone alteration with spodumene being replaced by micaceous minerals of muscovite, lepidolite and trilithionite, as well as calcite. In Pil2 and Pil3 samples the spodumene particles are generally free of mineral impurities except minor intergrowths of quartz, feldspar and spodumene are evident in the coarser fractions. Based on mineralogical observations in the current study, the majority of the main gangue minerals quartz, K feldspar and albite can be rejected at a coarse grind size of −4 mm, to recover 90% of the spodumene with Li upgrade from 0.99–1.5 wt% Li to 3.0–3.5 wt% (6.5–7.5 wt% Li 2 O). The iron content (81–1475 ppm) in the spodumene is low and therefore make these spodumene concentrates suitable for use in ceramic and glass applications. Recovery of spodumene in the coarse fractions could be improved by further particle size reduction to liberate spodumene from micas and feldspars in the middling class, which account for between 15 and 49% of the sample. However, the requirement to remove mineral impurities in the spodumene in downstream processing will be dependent on the method of processing as the presence of Li bearing micas, calcite and feldspar can be beneficial or detrimental to lithium recovery. The high content of Rb (1 wt%) and the abundance of free grains makes K feldspar a source of rubidium, particularly in the Pil3 sample which has K feldspar in high abundance (21 wt%) and can potentially be recovered by reverse flotation technique. The low concentrations of the Ta, Nb and Sn minerals identified in samples were found to be fairly well liberated and could be recovered by conventional gravity separation techniques

Mechanisms of base selection by human single-stranded selective monofunctional uracil-DNA glycosylase

hSMUG1 (human single-stranded selective monofunctional uracil-DNA glyscosylase) is one of three glycosylases encoded within a small region of human chromosome 12. Those three glycosylases, UNG (uracil-DNA glycosylase), TDG (thymine-DNA glyscosylase), and hSMUG1, have in common the capacity to remove uracil from DNA. However, these glycosylases also repair other lesions and have distinct substrate preferences, indicating that they have potentially redundant but not overlapping physiological roles. The mechanisms by which these glycosylases locate and selectively remove target lesions are not well understood. In addition to uracil, hSMUG1 has been shown to remove some oxidized pyrimidines, suggesting a role in the repair of DNA oxidation damage. In this paper, we describe experiments in which a series of oligonucleotides containing purine and pyrimidine analogs have been used to probe mechanisms by which hSMUG1 distinguishes potential substrates. Our results indicate that the preference of hSMUG1 for mispaired uracil over uracil paired with adenine is best explained by the reduced stability of a duplex containing a mispair, consistent with previous reports with Escherichia coli mispaired uracil-DNA glycosylase. We have also extended the substrate range of hSMUG1 to include 5-carboxyuracil, the last in the series of damage products from thymine methyl group oxidation. The properties used by hSMUG1 to select damaged pyrimidines include the size and free energy of solvation of the 5-substituent but not electronic inductive properties. The observed distinct mechanisms of base selection demonstrated for members of the uracil glycosylase family help explain how considerable diversity in chemical lesion repair can be achieved

RELICS: Strong Lens Models for Five Galaxy Clusters From the Reionization Lensing Cluster Survey

Strong gravitational lensing by galaxy clusters magnifies background galaxies, enhancing our ability to discover statistically significant samples of galaxies at z>6, in order to constrain the high-redshift galaxy luminosity functions. Here, we present the first five lens models out of the Reionization Lensing Cluster Survey (RELICS) Hubble Treasury Program, based on new HST WFC3/IR and ACS imaging of the clusters RXC J0142.9+4438, Abell 2537, Abell 2163, RXC J2211.7-0349, and ACT-CLJ0102-49151. The derived lensing magnification is essential for estimating the intrinsic properties of high-redshift galaxy candidates, and properly accounting for the survey volume. We report on new spectroscopic redshifts of multiply imaged lensed galaxies behind these clusters, which are used as constraints, and detail our strategy to reduce systematic uncertainties due to lack of spectroscopic information. In addition, we quantify the uncertainty on the lensing magnification due to statistical and systematic errors related to the lens modeling process, and find that in all but one cluster, the magnification is constrained to better than 20% in at least 80% of the field of view, including statistical and systematic uncertainties. The five clusters presented in this paper span the range of masses and redshifts of the clusters in the RELICS program. We find that they exhibit similar strong lensing efficiencies to the clusters targeted by the Hubble Frontier Fields within the WFC3/IR field of view. Outputs of the lens models are made available to the community through the Mikulski Archive for Space TelescopesComment: Accepted to Ap