Mapping site‐specific protein N‐glycosylations through liquid chromatography/mass spectrometry and targeted tandem mass spectrometry

Glycosylation is one of the most common posttranslational modifications (PTMs) of proteins, the characterization of which is commonly achieved through proteomic protocol, involving trypsin digestion followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, it is often not possible to characterize all glycopeptides in a complex sample because of the high complexity of glycoproteomic samples, and the relative lower abundances of glycopeptides in comparison to the unmodified peptides. We present here a targeted MS/MS analysis approach, which utilizes a previously developed computational tool, GlyPID, to guide multiple experiments, thus permitting a complete characterization of all N‐glycosylation sites of glycoproteins present in a complex sample. We have tested our approach using model glycoproteins analyzed by high‐resolution LTQ‐FT MS. The results demonstrate a potential use of our method for a high‐throughput characterization of complex mixtures of glycosylated proteins. Copyright © 2010 John Wiley & Sons, Ltd.     

Improving confidence in detection and characterization of protein N-glycosylation sites and microheterogeneity

Protein glycosylation is one of the most common post-translational modifications, estimated to occur in over 50% of human proteins. Mass spectrometry (MS)-based approaches involving different fragmentation mechanisms have been frequently used to detect and characterize protein N-linked glycosylations. In addition to the popular Collision-Induced Dissociation (CID), high-energy C-trap dissociation (HCD) fragmentation, which is a feature of a linear ion trap orbitrap hybrid mass spectrometer (LTQ Orbitrap), has been recently used for the fragmentation of tryptic N-linked glycopeptides in glycoprotein analysis. The oxonium ions observed with high mass accuracy in the HCD spectrum of glycopeptides can be combined with characteristic fragmentation patterns in the CID spectrum resulting from consecutive glycosidic bond cleavages, to improve the detection and characterization of N-linked glycopeptides. As a means of automating this process, we describe here GlypID 2.0, a software tool that implements several algorithmic approaches to utilize MS information including accurate precursor mass and spectral patterns from both HCD and CID spectra, thus allowing for an unequivocal and accurate characterization of N-linked glycosylation sites of proteins.     

Enzyme-free colorimetric assay of serum uric acid

A nonenzymatic method for the selective detection and quantification of serum uric acid (UA) using 2-thiouracil (2-TU) tailored Au nanoparticles is developed. The H-bonding interaction of UA with functionalized Au nanoparticles brings instantaneous visible color change and paves the way for the visible sensing of UA.     

Elastic scattering of electrons by metastable hydrogen atoms in the presence of a resonant laser field

Within the framework of the rotating wave approximation the elastic scattering of electrons by metastable 2s state of hydrogen atoms is studied in the presence of a resonant laser field. The frequency of the circularly polarized laser field is chosen to match the 2s-3p transition frequency in the hydrogen atom. Variation of the cross section with laser intensity and with incident electron energy (50-150 eV) is investigated. Received: 18 July 1997 / Received in final form: 5 December 1997 / Accepted: 19 January 1998     

Assessing outcomes for dysphonic patients

This study was designed to evaluate a disease-specific outcome measure for patients with selected voice disorders and to relate this instrument to a standardized quality of life measurement. In addition, the study attempts to document the degree of handicap for dysphonia patients globally, between different vocal pathologies, and in comparison to other chronic diseases. In this prospective, observational study, 260 adult patients evaluated for alterations of voice completed a general quality of life measure (the Medical Outcomes Trust Short Form 36-Item[SF-36]) and a voice-specific instrument (Voice Handicap Index [VHI]) pretreatment.

The highest correlation was between the social functioning score of the SF-36and the total score of the VHI and the physical, emotional, and functional subscales (p < 0.001) of the VHI. Significant correlation was also obtained for the SF-36 domains mental health (p < 0.01), general health (p < 0.01), and role functioning emotional (p < 0.017) with the three VHI domains and the total VHI score. Patients had significantly lower scores than the general U.S. population in five of the eight domains of SF-36. Patients with vocal fold paralysis had the highest level of pretreatment disability as measured on both the VHI and SF-36 among voice patients. The patients with dysphonia had a lower level of physical functioning than the patients with chronic sinusitis (p < 0.01), reflecting a greater handicap. In addition, the dysphonia group had lower levels of social functioning than the angina (p < 0.01) and sciatica (p < 0.01) groups and a lower score for mental health than the angina group (p < 0.01).

The SF-36 correlates with the VHI in the domains of social functioning,mental health, and role functioning emotional. The baseline handicap for voice disorders represents a significant disability even in comparison to conditions such as angina pectoris, sciatica, and chronic sinusitis.     

Colossal Stability of SiB11(BO)12−: An Implication as Potential Electrolyte in High-Voltage Alkali-ion Battery

High-voltage alkali metal-ion batteries (AMIBs) require a non-hazardous, low-cost, and highly stable electrolyte with a large operating potential and rapid ion conductivity. Here, we have reported a halogen-free high-voltage electrolyte based on SiB₁₁(BO)₁₂⁻. Because of the weak π-orbital interaction of −BO as well as the mixed covalent and ionic interaction between SiB₁₁-cage and −BO ligand, SiB₁₁(BO)₁₂⁻ has colossal stability. SiB₁₁(BO)₁₂⁻ possesses extremely high vertical detachment energy (9.95 eV), anodic voltage limit (∼10.05 V), and electrochemical stability window (∼9.95 V). Furthermore, SiB₁₁(BO)₁₂⁻ is thermodynamically stable at high temperatures, and its large size allows for faster cation movement. The alkali salts MSiB₁₁(BO)₁₂ (M=Li, Na, and K) are easily dissociated into ionic components. Electrolytes based on SiB₁₁(BO)₁₂⁻ greatly outperform commercial electrolytes. In short, SiB₁₁(BO)₁₂⁻-based compound is demonstrated to be a high-voltage electrolyte for AMIBs.     

A p‐Hydroxyphenacyl–Benzothiazole–Chlorambucil Conjugate as a Real‐Time‐Monitoring Drug‐Delivery System Assisted by Excited‐State Intramolecular Proton Transfer

Among the well‐known phototriggers, the p‐hydroxyphenacyl (pHP) group has consistently enabled the very fast, efficient, and high‐conversion release of active molecules. Despite this unique behavior, the pHP group has been ignored as a delivery agent, particularly in the area of theranostics, because of two major limitations: Its excitation wavelength is below 400 nm, and it is nonfluorescent. We have overcome these limitations by incorporating a 2‐(2′‐hydroxyphenyl)benzothiazole (HBT) appendage capable of rapid excited‐state intramolecular proton transfer (ESIPT). The ESIPT effect also provided two unique advantages: It assisted the deprotonation of the pHP group for faster release, and it was accompanied by a distinct fluorescence color change upon photorelease. In vitro studies showed that the p‐hydroxyphenacyl–benzothiazole–chlorambucil conjugate presents excellent properties, such as real‐time monitoring, photoregulated drug delivery, and biocompatibility.     

Fluorescence and biochemical characterization of glycated hemoglobin

Isoelectric focusing (IEF) of glycated hemoglobin (GHb) was carried out in ultra-thin polyacrylamide gels to separate the hemoglobin-advanced glycation endproducts (Hb-AGEs) from the hemoglobin-A_1C (HbA_1C) fraction. Precast polyacrylamide gels (Ampholine^® PAGplate) were used in Pharmacia LKB Multiphor II for this purpose. The separated bands for Hb-AGE and HbA_1C based on their isoelectric point (pI), were confirmed with the purifed fractions obtained from the cation exchange chromatographic technique. From the calibration curve, the pI values were found to be 6.748 and 6.495 for HbA_1C and Hb-AGE, respectively. The lowering of pI values for glycated hemoglobin, when compared to unglycated hemoglobin (pI = 6.852), can be attributed to the glycation at the amino terminals of the peptide chains. Increased reduction in pI value for Hb-AGE can be attributed to the effect of glycation of amino groups at various sites on the peptide chains, apart from the terminal amino groups. Fluorescence analysis was carried out for the purified fraction of Hb-AGE which showed the formation of a new fluorophor adduct having the excitation and emission maxima at 308 nm and 345 nm, respectively. Time-dependent formation of Hb-AGE under in vitro conditions was monitored by fluorescence (308/345 nm) over a period of 120 days, which showed its formation only after 3 weeks of incubation.