Variability in protein cargo detection in technical and biological replicates of exosome-enriched extracellular vesicles

Exosomes are extracellular vesicles (EVs) of ~20–200 nm diameter that shuttle DNAs, RNAs, proteins and other biomolecules between cells. The large number of biomolecules present in exosomes demands the frequent use of high-throughput analysis. This, in turn, requires t echnical r eplicate s (TRs), and b iological r eplicate s (BRs) to produce accurate results. As the number and abundance of identified biomolecules varies between r eplicate s (Rs), establishing the replicate variability predicted for the event under study is essential in determining the number of Rs required. Although there have been few reports of replicate variability in high throughput biological data, none of them focused on exosomes. Herein, we determined the replicate variability in protein profiles found in exosomes released from 3 lung adenocarcinoma cell lines, H1993, A549 and H1975. Since exosome isolates are invariably contaminated by a small percentage of ~200–300 nm microvesicles, we refer to our samples as e xosome- e nriched EVs (EE-EVs). We generated BRs of EE-EVs from each cell line, and divided each group into 3 TRs. All Rs were analyzed by l iquid c hromatography/ m ass s pectrometry (LC/MS/MS) and customized bioinformatics and biostatistical workflows (raw data available via ProteomeXchange: PXD012798). We found that the variability among TRs as well as BRs, was largely qualitative (protein present or absent) and higher among BRs. By contrast, the quantitative (protein abundance) variability was low, save for the H1975 cell line where the quantitative variability was significant. Importantly, our replicate strategy identified 90% of the most abundant proteins, thereby establishing the utility of our approach.