Protein Adsorption Loss─The Bottleneck of Single-Cell Proteomics.

Single-cell proteomics is a promising field to provide direct yet comprehensive molecular insights into cellular functions without averaging effects. Here, we address a grand technical challenge impeding the maturation of single-cell proteomics─protein adsorption loss (PAL). Even though widely known, there is currently no quantitation on how profoundly and selectively PAL has affected single-cell proteomics. Therefore, the mitigations to this challenge have been generic, and their efficacy was only evaluated by the size of the resolved proteome with no specificity on individual proteins. We use the existing knowledge of PAL, protein expression, and the typical surface area used in single-cell proteomics to discuss the severity of protein loss. We also summarize the current solutions to this challenge and briefly review the available methods to characterize the physical and chemical properties of protein surface adsorption. By citing successful strategies in single-cell genomics for measurement errors in individual transcripts, we pinpoint the urgency to benchmark PAL at the proteome scale with individual protein resolution. Finally, orthogonal single-cell proteomic techniques that have the potential to cross validate PAL are proposed. We hope these efforts can promote the fruition of single-cell proteomics in the near future.

Absolute Quantification of Nav1.5 Expression by Targeted Mass Spectrometry.

Nav1.5 is the pore forming α-subunit of the cardiac voltage-gated sodium channel that initiates cardiac action potential and regulates the human heartbeat. A normal level of Nav1.5 is crucial to cardiac function and health. Over- or under-expression of Nav1.5 can cause various cardiac diseases ranging from short PR intervals to Brugada syndromes. An assay that can directly quantify the protein amount in biological samples would be a priori to accurately diagnose and treat Nav1.5-associated cardiac diseases. Due to its large size (>200 KD), multipass transmembrane domains (24 transmembrane passes), and heavy modifications, Nav1.5 poses special quantitation challenges. To date, only the relative quantities of this protein have been measured in biological samples. Here, we describe the first targeted and mass spectrometry (MS)-based quantitative assay that can provide the copy numbers of Nav1.5 in cells with a well-defined lower limit of quantification (LLOQ) and precision. Applying the developed assay, we successfully quantified transiently expressed Nav1.5 in as few as 1.5 million Chinese hamster ovary (CHO) cells. The obtained quantity was 3 ± 2 fmol on the column and 3 ± 2 × 104 copies/cell. To our knowledge, this is the first absolute quantity of Nav1.5 measured in a biological sample.

Alternative splicing of ceramide synthase 2 alters levels of specific ceramides and modulates cancer cell proliferation and migration in Luminal B breast cancer subtype.

Global dysregulation of RNA splicing and imbalanced sphingolipid metabolism has emerged as promoters of cancer cell transformation. Here, we present specific signature of alternative splicing (AS) events of sphingolipid genes for each breast cancer subtype from the TCGA-BRCA dataset. We show that ceramide synthase 2 (CERS2) undergoes a unique cassette exon event specifically in Luminal B subtype tumors. We validated this exon 8 skipping event in Luminal B cancer cells compared to normal epithelial cells, and in patient-derived tumor tissues compared to matched normal tissues. Differential AS-based survival analysis shows that this AS event of CERS2 is a poor prognostic factor for Luminal B patients. As Exon 8 corresponds to catalytic Lag1p domain, overexpression of AS transcript of CERS2 in Luminal B cancer cells leads to a reduction in the level of very-long-chain ceramides compared to overexpression of protein-coding (PC) transcript of CERS2. We further demonstrate that this AS event-mediated decrease of very-long-chain ceramides leads to enhanced cancer cell proliferation and migration. Therefore, our results show subtype-specific AS of sphingolipid genes as a regulatory mechanism that deregulates sphingolipids like ceramides in breast tumors, and can be explored further as a suitable therapeutic target.

Structural similarity, characterization of Poly Ethylene Glycol linkage and identification of product related variants in biosimilar pegfilgrastim.

The approval of biosimilars requires demonstration of biosimilarity, which rests on the base of thorough analytical characterization of the biosimilar product. In addition to demonstration of biosimilarity, the product related impurities need to be thoroughly characterized and controlled at minimal levels. Pegylation of peptides and proteins creates significant challenges for detailed structural characterization, such as PEG (Poly Ethylene Glycol) heterogeneity, site of addition and number of attached pegylated moieties. A combination of several methods including circular dichroism, FTIR (Fourier-transform Infrared Spectroscopy), fluorescence spectroscopy, DSC (Differential Scanning Calorimetry), 1D and 2D NMR (Nuclear Magnetic Resonance), Edman degradation and peptide mapping by LC-MS (Liquid Chromatography Mass Spectrometry) were used for characterization of N-terminally pegylated filgrastim. Product related impurities such as oxidized, reduced, deamidated, dipegylated variants and monopegylated positional isomers have been characterized in detail using various HPLC (High Performance Liquid Chromatography) based methods and LC-MS techniques. The functional characterization in terms of receptor binding and cell proliferation assay was conducted for the similarity assessment and the potential impact of the product variants on the in vitro biological activity has also been assessed. In summary, this study presents, for the first time, a detailed structural and molecular level characterization of a biosimilar pegfilgrastim providing a strong base for the demonstration of overall biosimilarity of the product with the innovator product.

Oscillatory phenomena at liquid-liquid interfaces.

The mechanism of (i) Yoshikawa and Matsubara liquid-liquid interface oscillators and (ii) density/salt-water oscillators has been investigated. A modified simple mathematical formalism of both has been presented. Qualitative support for the model is provided by the available experimental results.