Chemical Derivatization of Peptide Carboxyl Groups for Highly Efficient Electron Transfer Dissociation

The carboxyl groups of tryptic peptides were derivatized with a tertiary or quaternary amine labeling reagent to generate more highly charged peptide ions that fragment efficiently by electron transfer dissociation (ETD). All peptide carboxyl groups—aspartic and glutamic acid side-chains as well as C-termini—were derivatized with an average reaction efficiency of 99 %. This nearly complete labeling avoids making complex peptide mixtures even more complex because of partially-labeled products, and it allows the use of static modifications during database searching. Alkyl tertiary amines were found to be the optimal labeling reagent among the four types tested. Charge states are substantially higher for derivatized peptides: a modified tryptic digest of bovine serum albumin (BSA) generates ~90% of its precursor ions with z? > ?2, compared with less than 40 % for the unmodified sample. The increased charge density of modified peptide ions yields highly efficient ETD fragmentation, leading to many additional peptide identifications and higher sequence coverage (e.g., 70 % for modified versus only 43 % for unmodified BSA). The utility of this labeling strategy was demonstrated on a tryptic digest of ribosomal proteins isolated from yeast cells. Peptide derivatization of this sample produced an increase in the number of identified proteins, a >50 % increase in the sequence coverage of these proteins, and a doubling of the number of peptide spectral matches. This carboxyl derivatization strategy greatly improves proteome coverage obtained from ETD-MS/MS of tryptic digests, and we anticipate that it will also enhance identification and localization of post-translational modifications.

Improved Identification and Relative Quantification of Sites of Peptide and Protein Oxidation for Hydroxyl Radical Footprinting

Protein oxidation is typically associated with oxidative stress and aging and affects protein function in normal and pathological processes. Additionally, deliberate oxidative labeling is used to probe protein structure and protein–ligand interactions in hydroxyl radical protein footprinting (HRPF). Oxidation often occurs at multiple sites, leading to mixtures of oxidation isomers that differ only by the site of modification. We utilized sets of synthetic, isomeric “oxidized” peptides to test and compare the ability of electron-transfer dissociation (ETD) and collision-induced dissociation (CID), as well as nano-ultra high performance liquid chromatography (nanoUPLC) separation, to quantitate oxidation isomers with one oxidation at multiple adjacent sites in mixtures of peptides. Tandem mass spectrometry by ETD generates fragment ion ratios that accurately report on relative oxidative modification extent on specific sites, regardless of the charge state of the precursor ion. Conversely, CID was found to generate quantitative MS/MS product ions only at the higher precursor charge state. Oxidized isomers having multiple sites of oxidation in each of two peptide sequences in HRPF product of protein Robo-1 Ig1-2, a protein involved in nervous system axon guidance, were also identified and the oxidation extent at each residue was quantified by ETD without prior liquid chromatography (LC) separation. ETD has proven to be a reliable technique for simultaneous identification and relative quantification of a variety of functionally different oxidation isomers, and is a valuable tool for the study of oxidative stress, as well as for improving spatial resolution for HRPF studies.

Amperometric Detection of Histamine with a Pyrroloquinoline‐Quinone Modified Electrode

A modified glassy carbon (GC) electrode was developed for the amperometric detection of biogenic amines, particularly histamine. The electrode was modified with the co‐enzyme pyrroloquinoline quinone (PQQ) by entrapment during electropolymerziation of pyrrole to form polypyrrole (PPy). This method formed a thin film on the electrode surface possessing very good stability with a shelf‐life exceeding one month without loss of signal. Optimal conditions for the PQQ/PPy electrode were determined and a linear response was found for histamine in phosphate buffer (pH 6) at +550 mV from 40 to 170 mg L^?1 with a limit of detection (S/N≥3) of 38 mg L^?1. The practical linear range offered by this method suggests ideal use for spoilage detection in fermented foods.     

Mixed-Isotope Labeling with LC-IMS-MS for Characterization of Protein–Protein Interactions by Chemical Cross-Linking

Chemical cross-linking of proteins followed by proteolysis and mass spectrometric analysis of the resulting cross-linked peptides provides powerful insight into the quaternary structure of protein complexes. Mixed-isotope cross-linking (a method for distinguishing intermolecular cross-links) was coupled with liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS) to provide an additional separation dimension to the traditional cross-linking approach. This method produced multiplet m/z peaks that are aligned in the IMS drift time dimension and serve as signatures of intermolecular cross-linked peptides. We developed an informatics tool to use the amino acid sequence information inherent in the multiplet spacing for accurate identification of the cross-linked peptides. Because of the separation of cross-linked and non-cross-linked peptides in drift time, our LC-IMS-MS approach was able to confidently detect more intermolecular cross-linked peptides than LC-MS alone.      

Separation of 1,3‐dimethylamylamine and other polar compounds in a dietary supplement formulation using aqueous normal phase chromatography with MS

Five ingredients (caffeine, l ‐arginine, creatine, β‐alanine, and 1,3‐dimethylamylamine) from a workout supplement were separated by HPLC with UV detection and LC–MS using an analytical column based on silica hydride operating in aqueous normal phase mode. While RP methods were observed to be inadequate for the analysis due to low retention, aqueous normal phase chromatography was able to readily retain and resolve the analytes. After method development on the HPLC–UV system, the conditions were successfully transferred to an LC–MS system for analysis. Based on calibration curve data, estimates of 63.5, 380.3, and 13.1 mg/serving (5.50 g) were obtained for creatine, l ‐arginine, and 1,3‐dimethylamylamine, respectively. Standard addition data results were compared to those of the calibration curve study, and the two values differed by less than 1% in the case of creatine. The conditions are suitable for further development as a reliable means of quantitating the analytes in workout supplement formulations.     

Distinguishing Ontario ginseng landraces and ginseng species using NMR-based metabolomics

The use of ¹H-NMR-based metabolomics to distinguish and identify unique markers of five Ontario ginseng (Panax quinquefolius L.) landraces and two ginseng species (P. quinquefolius and P. ginseng) was evaluated. Three landraces (2, 3, and 5) were distinguished from one another in the principal component analysis (PCA) scores plot. Further analysis was conducted and specific discriminating metabolites from the PCA loadings were determined. Landraces 3 and 5 were distinguishable on the basis of a decreased NMR intensity in the methyl ginsenoside region, indicating decreased overall ginsenoside levels. In addition, landrace 5 was separated by an increased amount of sucrose relative to the rest of the landraces. Landrace 2 was separated from the rest of the landraces by the increased level of ginsenoside R_b1. The Ontario P. quinquefolius was also compared with Asian P. ginseng by PCA, and clear separation between the two groups was detected in the PCA scores plot. The PCA loadings plot and a t-test NMR difference plot were able to identify an increased level of maltose and a decreased level of sucrose in the Asian ginseng compared with the Ontario ginseng. An overall decrease of ginsenoside content, especially ginsenoside R_b1, was also detected in the Asian ginseng’s metabolic profile. This study demonstrates the potential of NMR-based metabolomics as a powerful high-throughput technique in distinguishing various closely related ginseng landraces and its ability to identify metabolic differences from Ontario and Asian ginseng. The results from this study will allow better understanding for quality assessment, species authentication, and the potential for developing a fully automated method for quality control.

Microwave Synthesis of 5-Substituted 1H-Tetrazoles Catalyzed by Bismuth Chloride in Water

Bismuth chloride was used to catalyze the [2 + 3] cycloaddition between sodium azide with aryl nitriles, aliphatic nitriles, and vinyl nitriles. A number of 5-substituted 1H-tetrazoles were synthesized in water or isopropanol/water mixtures using microwave heating. Good yields were obtained for these reactions when heated for 1 h at 120–160 °C in a 3:1 isopropanol/water mixture. A few of the less reactive nitriles required longer reaction times for good yields.     

Data and device protection: A ToF‐SIMS,wetting, and XPS study of an Apple iPod nano

An important aspect of the robustness of an electronic device is its ability to resist water, fingerprints, dirt, and smudges that may compromise its ability to function and/or the information within it. Here, we report a chemical analysis by ToF‐SIMS, wetting, and XPS of the surfaces in a commercially available Apple iPod nano (8GB, MC525LL/A), which showed good resistance to its environment. This analysis reveals that the front panel (touchscreen) of the device is coated with a low free energy fluorinated polymer that may consist of short segments of a fluorinated hydrocarbon connected through ether linkages. No other part of the device appears to have this hydrophobic coating. A plasma treatment of the device leads to a deterioration of its performance. This work demonstrates how different analytical techniques can complement each other and contribute to a better understanding of a surface or a material. Copyright © 2013 John Wiley & Sons, Ltd.