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Arginine methylation has been recognized as a post-translational modification with pleiotropic effects that span from regulation of transcription to metabolic processes that contribute to aberrant cell proliferation and tumorigenesis. This has brought significant attention to the development of therapeutic strategies aimed at blocking the activity of protein arginine methyltransferases (PRMTs), which catalyze the formation of various methylated arginine products on a wide variety of cellular substrates. GSK3368715 is a small molecule inhibitor of type I PRMTs currently in clinical development. Here, we evaluate the effect of type I PRMT inhibition on arginine methylation in normal human peripheral blood mononuclear cells and utilize a broad proteomic approach to identify type I PRMT substrates. This work identified heterogenous nuclear ribonucleoprotein A1 (hnRNP-A1) as a pharmacodynamic biomarker of type I PRMT inhibition. Utilizing targeted mass spectrometry (MS), methods were developed to detect and quantitate changes in methylation of specific arginine residues on hnRNP-A1. This resulted in the development and validation of novel MS and immune assays useful for the assessment of GSK3368715 induced pharmacodynamic effects in blood and tumors that can be applied to GSK3368715 clinical trials.

The structural and functional versatility of immunoglobulinG (IgG) scaffold of mAbs also facilitates the design of more complex compounds, such as antibody-drug conjugates, Fc-fusion proteins and bispecific antibodies (2). Since the functions of mAb-based biotherapeutics are typically achieved by protein-protein or protein-ligand interactions, direct analysis of such noncovalent interactions has great potential to elucidate the pharmacology and toxicology of mAbs. [...]to obtain the information of noncovalent macromolecular assemblies (such as ligand binding, substrate turnover and subunit stoichiometry), native MS has attracted increasing research interest (11,12). [...]the number of charges associated with the protein is usually reduced compared with intact MS analysis, indicating a more compact and folded structure under the native conformation. Because the integrity of proteins is not disrupted, native MS provides a wealth of information for understanding biological properties of intact protein assemblies under near-physiological conditions. The major requirement for buffer in ESI is volatility, which ensures the analyte completely transferring into the gas phase and reduces the formation of adducts with nonvolatile salts impacting ion signals (16). [...]a compatible buffer is also necessary for native MS. Buffer with strong acidity and significant amount of organic solvent can open the native conformation of protein to a less compacted partially folded form and hereby should be avoided (17).

Recent advances in microflow ultra performance liquid chromatography (UPLC) systems offer higher sensitivity with robustness to meet the routine bioanalytical demands. Modern high-resolution mass spectrometers (HRMS) enable the development of highly selective methods with broad dynamic range. The quantitative performances of tandem quadrupole MS and HRMS were comprehensively compared using seven intact peptide hormones up to 9.4 kDa. Results show comparable performance between two platforms in sensitivity, accuracy and linearity. For some peptides, HRMS provided lower background interference. The benefit of increased sensitivity using microflow UPLC was also demonstrated. HRMS is a versatile platform capable of both basic characterization and reliable quantitation in complex matrices. Microflow UPLC provides lower LLOQs than conventional flow systems, even with less sample volume injected.

In the pharmaceutical industry, proteomics has long been utilized as a drug-discovery tool to help understand changes in protein profiles for disease states or protein expression in relation to genomic studies for target discovery or identification (1). Current proteomics platforms Affinity-based platforms to perform quantitation of protein biomarkers to support clinical studies utilize either antibodies (e.g., O-Myriad, Kiloplex1000) or aptamers (e.g., Somascan) to capture the proteins of interest with quantitation varying from traditional immunoassay to array-based quantitation. Focusing more toward application (as opposed to development), a need exists for experts in biological systems partnered with trained analytical scientists as a two-way mentorship of sorts, to maximize potential gains when performing global sample analysis. Fractionation at the peptide level (after enzymatic digestion) has become the most common approach to decrease sample complexity for proteomic experiments. 2D LC separation, with low-pH reverse phase as the second dimension, is a common strategy to fractionate a peptide mixture prior to tandem MS analysis.

Antibody biotherapeutic measurement from pharmacokinetic studies has not been traditionally based on intact molecular mass as is the case for small molecules. However, recent advancements in protein capture and mass spectrometer technology have enabled intact mass detection and quantitation for dosed biotherapeutics. A bioanalytical method validation is part of the regulatory requirement for sample analysis to determine drug concentration from in-life study samples. Here, an intact protein LC–MS assay is subjected to mock bioanalytical method validation, and unknown samples are compared between intact protein LC–MS and established bioanalytical assay formats: Ligand-binding assay and peptide LC–MS/MS. Results are presented from the intact and traditional bioanalytical method evaluations, where the in-life sample concentrations were comparable across method types with associated data analyses presented. Furthermore, for intact protein LC–MS, modification monitoring and evaluation of data processing parameters is demonstrated.

The ability to quantitate small molecule therapeutics using dried blood spot (DBS) sampling has been driven in the pharmaceutical industry by its promise of reducing and refining the use of study animals in toxicokinetic studies. In addition, DBS techniques have been of interest in a clinical setting as a way to deliver a less invasive sampling method without the need for freezer storage of the samples. In the last few years there has been some interest in extending the use of DBS from just small molecule drugs to peptide and protein therapeutics. This chapter gives an overview of recent literature and provides commentary on the strengths and weaknesses associated with the use of the DBS sampling technique for quantitative large molecule protein bioanalytical study support using either immunoassay or LC‐MS/MS methodologies.