Defining the Soluble and Extracellular Vesicle Protein Compartments of Plasma Using In-Depth Mass Spectrometry-Based Proteomics.

Plasma-derived extracellular vesicles (pEVs) are a potential source of diseased biomarker proteins. However, characterizing the pEV proteome is challenging due to its relatively low abundance and difficulties in enrichment. This study presents a streamlined workflow to identify EV proteins from cancer patient plasma using minimal sample input.

Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin.

Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co-isolated entities, a combination of methods is needed. However, this is time-consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available.

Evaluation of Spin Columns for Human Plasma Depletion to Facilitate MS-Based Proteomics Analysis of Plasma.

High abundant protein depletion is a common strategy applied to increase analytical depth in global plasma proteomics experiment setups. The standard strategies for depletion of the highest abundant proteins currently rely on multiple-use HPLC columns or multiple-use spin columns. Here we evaluate the performance of spin columns for plasma depletion and show that the single-use spin reduces handling time by allowing parallelization and is easily adapted to a nonspecialized lab environment without reducing the high plasma proteome coverage and reproducibility.

Single-Stranded Nucleic Acids Regulate TLR3/4/7 Activation through Interference with Clathrin-Mediated Endocytosis.

Recognition of nucleic acids by endosomal Toll-like receptors (TLR) is essential to combat pathogens, but requires strict control to limit inflammatory responses. The mechanisms governing this tight regulation are unclear. We found that single-stranded oligonucleotides (ssON) inhibit endocytic pathways used by cargo destined for TLR3/4/7 signaling endosomes.

Quantitative accuracy in mass spectrometry based proteomics of complex samples: the impact of labeling and precursor interference.

Unlabelled: Knowing the limit of quantification is important to accurately judge the results from proteomics studies. In order to investigate isobaric labels in combination with peptide pre-fractionation by high resolution isoelectric focusing in terms of limit of detection, quantitative accuracy and how to improve it, we used a human cell lysate spiked with 57 protein standards providing reference points across a wide concentration range. Specifically, the impact of precursor mixing (isolation interference and reporter ion interference) on quantitative accuracy was investigated by co-analyzing iTRAQ (8-plex) and TMT (6-plex) labeled peptides.

Gender differences in the bronchoalveolar lavage cell proteome of patients with chronic obstructive pulmonary disease.

Background: Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide and is increasing, primarily among women. Underdiagnosis is common, and because of the heterogeneous disease characteristics, molecular markers of specific disease phenotypes and more efficacious treatment regimens are urgently needed.

Objective: In this study the soluble proteome of bronchoalveolar lavage cells, primarily consisting of macrophages, was investigated with the aim of identifying phenotypic differences in early disease development.

Assessing recent smoking status by measuring exhaled carbon monoxide levels.

Background: Cigarette smoke causes both acute and chronic changes of the immune system. Excluding recent smoking is therefore important in clinical studies with chronic inflammation as primary focus. In this context, it is common to ask the study subjects to refrain from smoking within a certain time frame prior to sampling.

Fluorescence cross-correlation spectroscopy of a pH-sensitive ratiometric dye for molecular proton exchange studies.

Fluorescence fluctuation analysis of individual pH-sensitive fluorophores has recently proven to be a useful approach for biomolecular proton exchange studies. In this work, dual-color fluorescence cross-correlation spectroscopy (FCCS) is demonstrated on a ratiometric pH-sensitive dye, for which both the excitation and emission spectra shift as a function of pH. In the FCCS measurements, the fluorescence signal from the predominant emission wavelength range of the protonated form of the dye is cross-correlated with that of the deprotonated form.