Metabolite identification and quantitation of RBD1016 siRNA: a direct comparison of hybridization-based LC-FD and LC-HRAM assays.

RBD1016 is an -acetylgalactosamine-conjugated siRNA drug currently in a phase II trial for treatment of chronic hepatitis B virus. To evaluate its absorption, distribution, metabolism and excretion (ADME) and pharmacokinetic/pharmacodynamic (PK/PD) properties, two LC-based bioanalytical methods, LC-high-resolution/accuracy MS and LC-fluorescence detection, were developed and qualified. The LC-high-resolution/accuracy MS method was used for metabolite identification and simultaneous quantitation of the antisense and sense strands as well as their respective metabolites.

Lithium storage performance enhanced by lithiation-induced structural phase transitions of fluorinated MXenes.

Structural phase transitions in electrode materials of Li-ion batteries (LIBs) often occur along with Li-ion extraction/intercalation during charge and discharge processes. Lithiation-induced phase transition behaviors of two-dimensional fluorinated MXenes were investigated systematically by first-principles density functional calculations. The calculated results show that fluorine atoms in the nine MXenes studied moved from the FCC site (or HCP site for TaCF) to the TOP site during Li adsorption.

Regulated bioanalysis of liposomal amphotericin B to support pharmacokinetic studies of liposomal drugs.

Because of the delicate nature of liposomes, bioanalysis of free and liposomal-encapsulated drugs is among the most challenging assays to perform. Current regulatory guidance for bioanalysis is not sufficient to address the complexity of this particular formulation. Three individual LC-MS/MS methods to quantify free amphotericin B (10-3000 ng/ml) and encapsulated amphotericin B (100-50,000 ng/ml) in pretreated human plasma and total amphotericin B (100-50,000 ng/ml) in human plasma were fully validated and applied to a bioequivalence study.

Direct quantitation of free, encapsulated, total doxorubicin and doxorubicinol in stabilized frozen human plasma to support a BE study of liposomal doxorubicin.

Regulatory guidance requires the quantification of encapsulated and free doxorubicin for a liposomal doxorubicin injection bioequivalence study. Due to the instability of liposome formulations in plasma samples, the release of free drug from the liposomal encapsulated doxorubicin during sample handling would result in elevation of measured free doxorubicin concentration. To prevent the potential release of free drug, stabilizer reagents and procedures were successfully developed and validated to adequately stabilize liposomal drugs in plasma samples during sample collection, storage and extraction.

Importance of probe design for bioanalysis of oligonucleotides using hybridization-based LC-fluorescence assays.

The importance of the length and/or structure of fluorescently labeled PNA (peptide nucleic acid) probes for quantitative determination of oligodeoxynucleotides (ODNs) is demonstrated in human plasma using hybridization-based LC-fluorescence assays. The length of the PNA probes impacts the peak shape and chromatographic separation of the resulting PNA/ODN hybridization complexes and affects assay sensitivity, dynamic range and carryover. For quantitative determination of an 18-mer phosphodiester ODN (DNL1818) in human plasma, an assay utilizing an Atto dye-labeled 12-mer PNA probe provided a linear quantitation range of 0.

Direct Analysis of Protein S-Acylation by Mass Spectrometry.

Dynamic and reversible protein S-acylation, most commonly occurring as S-palmitoylation, plays an important role in protein/membrane association and the regulation of intracellular signaling via cycles of palmitoylation and depalmitoylation. Direct analysis of protein S-acylation by mass spectrometry (MS) offers several benefits over indirect detection methods in that it can definitively determine the location and nature of the acyl modification, and is not prone to false discoveries. However, characterization of acyl proteins is challenging because of the tendency of acyl loss during sample preparation and tandem MS analysis.

S- to N-Palmitoyl Transfer During Proteomic Sample Preparation.

N-palmitoylation has been reported in a number of proteins and suggested to play an important role in protein localization and functions. However, it remains unclear whether N-palmitoylation is a direct enzyme-catalyzed process, or results from intramolecular S- to N-palmitoyl transfer. Here, using the S-palmitoyl peptide standard, GCpalmLGNAK, as the model system, we observed palmitoyl migration from the cysteine residue to either the peptide N-terminus or the lysine side chain during incubation in both neutral and slightly basic buffers commonly used in proteomic sample preparation.

Surfactant-Induced Artifacts during Proteomic Sample Preparation.

Bottom-up proteomics is a powerful tool for characterization of protein post-translational modifications (PTMs), where PTMs are identified at the peptide level by mass spectrometry (MS) following protein digestion. However, enzymatic digestion is associated with additional sample processing steps that may potentially introduce artifactual modifications. Here, during an MS study of the PTMs of the regulator of G-protein signaling 4, we discovered that the use of ProteaseMAX, which is an acid-labile surfactant commonly used to improve protein solubilization and digestion efficiency, can lead to in vitro modifications on cysteine residues.

Direct detection of S-palmitoylation by mass spectrometry.

Direct detection and quantification of protein/peptide palmitoylation by mass spectrometry (MS) is a challenging task because of the tendency of palmitoyl loss during sample preparation and tandem MS analysis. In addition, the large difference in hydrophobicity between the palmitoyl peptides and their unmodified counterparts could prevent their simultaneous analysis in a single liquid chromatography-MS experiment. Here, the stability of palmitoylation in several model palmitoyl peptides under different incubation and fragmentation conditions was investigated.