flippant-An R package for the automated analysis of fluorescence-based scramblase assays.

BACKGROUND: The lipid scrambling activity of protein extracts and purified scramblases is typically measured using a fluorescence-based assay. While the assay has yielded insight into the scramblase activity in crude membrane preparations, functional validation of candidate scramblases, stoichiometry of scramblase complexes as well as ATP-dependence of flippases, data analysis in its context has remained a task involving many manual steps. RESULTS: With the extension package "flippant" to R, a free software environment for statistical computing and graphics, we introduce an integrated solution for the analysis and publication-grade graphical presentation of dithionite scramblase assays and demonstrate its utility in revisiting an originally manual analysis from the publication record, closely reproducing the reported results. CONCLUSIONS: "flippant" allows for quick, reproducible data analysis of scramblase activity assays and provides a platform for review, dissemination and extension of the strategies it employs.

Connecting genetic risk to disease end points through the human blood plasma proteome.

Genome-wide association studies (GWAS) with intermediate phenotypes, like changes in metabolite and protein levels, provide functional evidence to map disease associations and translate them into clinical applications. However, although hundreds of genetic variants have been associated with complex disorders, the underlying molecular pathways often remain elusive. Associations with intermediate traits are key in establishing functional links between GWAS-identified risk-variants and disease end points. Here we describe a GWAS using a highly multiplexed aptamer-based affinity proteomics platform. We quantify 539 associations between protein levels and gene variants (pQTLs) in a German cohort and replicate over half of them in an Arab and Asian cohort. Fifty-five of the replicated pQTLs are located in trans. Our associations overlap with 57 genetic risk loci for 42 unique disease end points. We integrate this information into a genome-proteome network and provide an interactive web-tool for interrogations. Our results provide a basis for novel approaches to pharmaceutical and diagnostic applications.

Complementarity of SOMAscan to LC-MS/MS and RNA-seq for quantitative profiling of human embryonic and mesenchymal stem cells.

Dynamic range limitations are challenging to proteomics, particularly in clinical samples. Affinity proteomics partially overcomes this, yet suffers from dependence on reagent quality. SOMAscan, an aptamer-based platform for over 1000 proteins, avoids that issue using nucleic acid binders. Targets include low expressed proteins not easily accessible by other approaches. Here we report on the potential of SOMAscan for the study of differently sourced mesenchymal stem cells (MSC) in comparison to LC-MS/MS and RNA sequencing. While targeting fewer analytes, SOMAscan displays high precision and dynamic range coverage, allowing quantification of proteins not measured by the other platforms. Expression between cell types (ESC and MSC) was compared across techniques and uncovered the expected large differences. Sourcing was investigated by comparing subtypes: bone marrow-derived, standard in clinical studies, and ESC-derived MSC, thought to hold similar potential but devoid of inter-donor variability and proliferating faster in vitro. We confirmed subtype-equivalency, as well as vesicle and extracellular matrix related processes in MSC. In contrast, the proliferative nature of ESC was captured less by SOMAscan, where nuclear proteins are underrepresented. The complementary of SOMAscan allowed the comprehensive exploration of CD markers and signaling molecules, not readily accessible otherwise and offering unprecedented potential in subtype characterization. SIGNIFICANCE: Mesenchymal stem cells (MSC) represent promising stem cell-derived therapeutics as indicated by their application in >500 clinical trials currently registered with the NIH. Tissue-derived MSC require invasive harvesting and imply donor-to-donor differences, to which embryonic stem cell (ESC)-derived MSC may provide an alternative and thus warrant thorough characterization. In continuation of our previous study where we compared in depth embryonic stem cells (ESC) and MSC from two sources (bone marrow and ESC-derived), we included the aptamer-based SOMAscan assay, complementing LC-MS/MS and RNA-seq data. Furthermore, SOMAscan, a targeted proteomics platform developed for analyzing clinical samples, has been benchmarked against established analytical platforms (LC-MS/MS and RNA-seq) using stem cell comparisons as a model.

readat: An R package for reading and working with SomaLogic ADAT files.

BACKGROUND: SomaLogic's SOMAscan™ assay platform allows the analysis of the relative abundance of over 1300 proteins directly from biological matrices such as blood plasma and serum. The data resulting from the assay is provided in a proprietary text-based format not easily imported into R. RESULTS: readat is an R package for working with the SomaLogic ADAT file format. It provides functionality for importing, transforming and annotating data from these files. The package is free, open source, and available on Bioconductor and Bitbucket. CONCLUSIONS: readat integrates into both Bioconductor and traditional R workflows, rendering it easy to make use of ADAT files.

Comprehensive transcriptomic and proteomic characterization of human mesenchymal stem cells reveals source specific cellular markers.

Mesenchymal stem cells (MSC) are multipotent cells with great potential in therapy, reflected by more than 500 MSC-based clinical trials registered with the NIH. MSC are derived from multiple tissues but require invasive harvesting and imply donor-to-donor variability. Embryonic stem cell-derived MSC (ESC-MSC) may provide an alternative, but how similar they are to ex vivo MSC is unknown. Here we performed an in depth characterization of human ESC-MSC, comparing them to human bone marrow-derived MSC (BM-MSC) as well as human embryonic stem cells (hESC) by transcriptomics (RNA-seq) and quantitative proteomics (nanoLC-MS/MS using SILAC). Data integration highlighted and validated a central role of vesicle-mediated transport and exosomes in MSC biology and also demonstrated, through enrichment analysis, their versatility and broad application potential. Particular emphasis was placed on comparing profiles between ESC-MSC and BM-MSC and assessing their equivalency. Data presented here shows that differences between ESC-MSC and BM-MSC are similar in magnitude to those reported for MSC of different origin and the former may thus represent an alternative source for therapeutic applications. Finally, we report an unprecedented coverage of MSC CD markers, as well as membrane associated proteins which may benefit immunofluorescence-based applications and contribute to a refined molecular description of MSC.

Comparison of urinary thallium levels in non-occupationally exposed people and workers.

PURPOSE: To determine a reference background urinary thallium level; to compare urinary thallium data from workers to this background level; to investigate factors affecting these levels and whether creatinine correction is appropriate. METHODS: Urine samples from non-occupationally exposed people (n = 273, from 113 individuals) and workers (n = 896, from 447 individuals) were analysed for thallium by ICP-MS. A reference background level was calculated, defined as the 95th percentile value of a non-occupationally exposed population. Worker data were divided into two subsets: thallium workers (those who work directly with thallium or its compounds) and general workers; and compared to the background level. Bayesian linear mixed effects modelling was used to investigate factors affecting urinary thallium concentration and the efficacy of creatinine correction for the determination of urinary thallium. RESULTS: The reference background urinary thallium level is 0.27 µmol/mol creatinine (creatinine-corrected) or 0.40 µg/l (uncorrected). Median values were 0.11 µmol/mol creatinine or 0.17 µg/l for non-occupationally exposed people, 0.12 µmol/mol creatinine or 0.20 µg/l for general workers and 0.19 µmol/mol creatinine or 0.41 µg/l for thallium workers. Variation was lower in creatinine-corrected models. Nine per cent of samples from general workers and 39 % of samples from thallium workers exceeded the creatinine-corrected background level. By 2010, 90 % of all workers had urinary thallium levels below the 95th percentile reference background level. CONCLUSIONS: Urinary thallium concentrations were higher in thallium workers than non-occupationally exposed people and general workers. Creatinine correction is appropriate.