JuliaLaskinChavaLifshitzPrinciples of Mass Spectrometry Applied to Biomolecules2006John Wiley & Sons, IncHoboken, NJ978-0-471-72184-007030, Hardcover, $150.00, 687 pp. ISBN:

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Ferrichrome: Surprising stability of a cyclic peptide-FeIII complex revealed by mass spectrometry

Ferrichrome, a fungal siderophore that is also utilized by some bacterial species, was studied with liquid secondary ion mass spectrometry (LSIMS) and matrix-assisted laser desorption ionixation (MALDI) mass spectrometry. A strong ionic signal corresponding to a FeIII complex was observed with LSIMS in the positive ion mode. Switching the polarity of the mass spectrometer did not necessarily result in reduction of ferric ion, although certain conditions led to appearance of a FeII complex signal as well. The results of the structural studies of the metal ion-cyclic peptide complex with collisionally induced dissociation allowed unambiguous identification of the chelation sites. The action of the siderophore on FeIII was studied by in vitro chelation of ferric ion (from ferric citrate) by the iron-free ferrichrome. Effective chelation of ferric ion was compared to actions of the iron-free ferrichrome on other metal ions. Unlike LSIMS, desorption with MALDI did not form selectively molecular ions of intact ferrichrome: the spectra contained abundant peaks corresponding to the cyclic peptide itself and its nonspecific association with alkali metal ions

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances

Mass Spectrometry in Biophysics

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Mass Spectrometry In Biophysics

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An ¹⁸O‑Labeling Assisted LC/MS Method for Assignment of Aspartyl/Isoaspartyl Products from Asn Deamidation and Asp Isomerization in Proteins

An ¹⁸O-labeling assisted LC/MS method was designed for unambiguous assignment of aspartyl/isoaspartyl products produced by Asn deamidation and Asp isomerization. By preparing the acid- and base-catalyzed deamidation standards in H₂¹⁸O, isomer-specific mass tags were introduced to aspartyl- and isoaspartyl-containing peptides, which could be easily distinguished by mass spectrometry (MS). In contrast to the traditional ways of assigning the isomers on the basis of their elution order in reverse phase HPLC, the new method is more reliable and universal. Furthermore, the new method can be applied to the entire protein digest, and is therefore more time- and cost-effective compared with existing methods that use synthetic aspartyl- and isoaspartyl-containing peptide standards. Finally, since the identification of isomers in the new method only relies on LC/MS analysis, it can be easily implemented using the most basic and inexpensive MS instrumentation, thus providing an attractive alternative to tandem MS based approaches. The feasibility of this new method is demonstrated using a model peptide as well as the entire digest of human serum transferrin

Overcoming the Hydrolytic Lability of a Reaction Intermediate in Production of Protein/Drug Conjugates: Conjugation of an Acyclic Nucleoside Phosphonate to a Model Carrier Protein

Acquired immunodeficiency syndrome (AIDS) remains one of the most serious public health challenges and a significant cause of mortality for certain populations. Despite the large number of antiretrovirals developed in the past two decades, the inability of small molecule therapeutics to target HIV reservoirs directly creates a significant obstacle to their effective utilization. Indeed, achieving the desired therapeutic outcome in the absence of an effective means of targeted delivery must rely on dosage escalation, which frequently causes severe toxicity. This problem may be solved by conjugation of antiretroviral agents to endogenous proteins that are specifically recognized by HIV reservoirs (such as macrophages) for internalization and catabolism. However, conjugation of a large class of antiretroviral agents (acyclic nucleoside phosphonates, such as adefovir, tenofovir, and cidofovir) to a protein is challenging due to rapid degradation of the activated form of the drug (e.g., adefovir phosphonoimidazolide) in an aqueous environment. A novel synthetic strategy introduced in this work overcomes the instability of the activated form of adefovir by emulating the first step of its metabolic pathway (phosphorylation), making it highly reactive toward primary amine groups of proteins and, at the same time, less prone to hydrolysis by water. Efficient conjugation of the phosphorylated form of adefovir to a protein following activation with EDC (1-ethyl-3-(3-(dimethylamino)­propyl)­carbodiimide hydrochloride) and imidazole was demonstrated using a model protein. Mass spectrometry (MS) was used to identify conditions that favor formation of conjugates with minimal side products, and online ion exchange chromatography/MS analysis of the products revealed the presence of multiple positional isomers within the 1:1 protein/drug conjugates. Both liquid chromatography/MS data and the analysis of ions produced upon top-down fragmentation of the 1:1 conjugates suggest that conjugation of phosphorylated adefovir to the protein occurs not only at primary amines but also at hydroxyl groups. The new conjugation protocol can be used to attach adefovir and other acyclic nucleoside phosphonates to proteins recognized by the cell surface receptors specific to macrophages (such as the haptoglobin/hemoglobin complex), enabling targeted drug delivery directly to HIV reservoirs