Strategies employing non-gel based methods for quantitative proteomic profiling such as isotope coded affinity tags coupled with mass spectrometry (ICAT-MS) are gaining attention as alternatives to two-dimensional gel electrophoresis (2-DE). We have conducted a large-scale investigation to determine the degree of reproducibility and depth of proteome coverage of a typical ICAT-MS experiment by measuring protein changes in Escherichia coli treated with triclosan, an inhibitor of fatty acid biosynthesis. The entire ICAT-MS experiment was conducted on four independent occasions where more than 24 000 peptides were quantitated using an ion-trap mass spectrometer. Our results demonstrated that quantitatively, the technique provided good reproducibility (median coefficient of variation of ratios was 18.6%), and on average identified more than 450 unique proteins per experiment. However, the method was strongly biased to detect acidic proteins (pI < 7), under-represented small proteins (<10 kDa) and failed to show clear superiority over 2-DE methods in monitoring hydrophobic proteins from cell lysates.

A new UV-filter compound, 4-(2-a~o-3-hy~oxyphenyl)~~xobu~noic acid O-glucoside, has been identified in human lenses. The structure suggests that it is derived biosynthetically from tryptophan. Quantification studies on the new compound show that it is the second-most abundant W-filter compound in the lens with an absorption and fluorescence spectrum similar to that of 3-hydroxykynurenine glucoside.

Recent findings suggest that the inhibition of Aurora A (AURKA) kinase may offer a novel treatment strategy against metastatic cancers. In the current study, we determined the effects of AURKA inhibition by the small molecule inhibitor MLN8237 both as a monotherapy and in combination with the microtubule targeting drug eribulin on different stages of metastasis in triple negative breast cancer (TNBC) and defined the potential mechanism of its action. MLN8237 as a single agent and in combination with eribulin affected multiple steps in the metastatic process including migration, attachment, and proliferation in distant organs, resulting in suppression of metastatic colonization and recurrence of cancer. Eribulin application induces accumulation of active AURKA in TNBC cells providing foundation for the combination therapy. Mechanistically, AURKA inhibition induced cytotoxic autophagy via activation of the LC3B/p62 axis and inhibition of pAKT, leading to eradication of metastases, but has no effect on growth of mammary tumor. Combination of MLN8237 with eribulin leads to a synergistic increase in apoptosis in mammary tumors, as well as cytotoxic autophagy in metastases. This preclinical data provides a new understanding of the mechanisms by which MLN8237 mediates its anti-metastatic effects and advocates for its combination with eribulin in future clinical trials for metastatic breast cancer and early stage solid tumors.

Depressed sarcoplasmic reticulum (SR) Ca-cycling is a hallmark of human and experimental heart failure. Strategies to improve this impairment by either increasing SERCA2a levels or decreasing phospholamban (PLN) activity have been suggested as promising therapeutic targets. Indeed, ablation of PLN gene in mice was associated with greatly enhanced cardiac Ca-cycling and performance. Intriguingly, this hyperdynamic cardiac function was maintained throughout the lifetime of the mouse without observable pathological consequences. To determine the cellular alterations in the expression or modification of myocardial proteins, which are associated with the enhanced cardiac contractility, we performed a proteomics-based analysis of PLN knockout (PLN-KO) hearts in comparison to isogenic wild-types. By use of 2-dimensional gel electrophoresis (2-DE), approximately 3300 distinct protein spots were detected in either wild-type or PLN-KO ventricles. Protein spots observed to be altered between PLN-KO and wild-type hearts were subjected to tryptic peptide mass fingerprinting for identification by MALDI-TOF mass spectrometry in combination with LC/MS/MS analysis. In addition, two-dimensional 32P-autoradiography was performed to analyze the phosphorylation profiles of PLN-KO cardiomyocytes. We identified alterations in the expression level of more than 100 ventricular proteins, along with changes in phosphorylation status of important regulatory proteins in the PLN-KO. These protein changes were observed mainly in two subcellular compartments: the cardiac contractile apparatus, and metabolism/energetics. Our findings suggest that numerous alterations in protein expression and phosphorylation state occurred upon ablation of PLN and that a complex functional relationship among proteins involved in calcium handling, myofibrils, and energy production may exist to coordinately maintain the hyperdynamic cardiac contractile performance of the PLN-KO mouse in the long term.

There is a growing industry and regulatory need to detect host cell protein (HCP) impurities in the production of protein biopharmaceuticals, as certain HCPs can impact product stability, safety, and efficacy, even at low levels. In some cases, regulatory agencies require the identification and the quantification of HCPs in drug products (DPs) for risk assessment, and this is an active and growing topic of conversation in the industry and amongst regulators. In this study, we developed a sensitive, robust, and reproducible workflow for HCP detection and quantification in a significantly shorter turnaround time than that previously reported using an Evosep ONE LC system coupled to an Orbitrap Fusion Lumos mass spectrometer. Because of its fast turnaround time, this HCP workflow can be integrated into process development for the high-throughput (60 samples analyzed per day) identification of HCPs. The ability to rapidly measure HCPs and follow their clearance throughout the downstream process can be used to pinpoint sources of HCP contamination, which can be used to optimize biopharmaceutical production to minimize HCP levels. Analysis of the NIST monoclonal antibody reference material using the rapid HCP profiling workflow detected the largest number of HCPs reported to date, underscoring an improvement in performance along with an increased throughput. The HCP workflow can be readily implemented and adapted for different purposes to guide biopharmaceutical process development and enable better risk assessment of HCPs in drug substances and DPs.