Comparative Proteomics of Unactivated and Activated Peripheral Blood Eosinophils

Introduction

Eosinophils play important roles in tissue homeostasis and the inflammation of allergic diseases such as asthma. Concomitant with extravasation into inflamed tissues, blood eosinophils are activated to undergo shape change and relocalization of surface receptors. A comprehensive knowledge of the eosinophil proteome, which to date has been limited to only a few hundred proteins, will be required to understand such activation. Here we report a draft map of the eosinophil proteome of unactivated peripheral blood eosinophils and characterize proteomic perturbations that develop upon acute activation by IL5, one of the major cytokines inducing migration of eosinophils into the lung in asthmatics.

Methods

For the global analysis of unactivated eosinophils, blood eosinophils purified from three individuals were combined, lysed by probe sonication, digested with trypsin, and desalted. The sample was enriched for phosphorylation using immobilized metal affinity chromatography (IMAC). The enriched and non-enriched samples were pre-fractionated using high pH reversed-phase chromatography. The fractions were analyzed on a nanoLC coupled to an Orbitrap Fusion (Thermo Scientific). MaxQuant was used to search, filter to 1% false discovery rate (FDR), and provide label free quantitation. Phosphorylation sites were localized with the PhosphoRS algorithm.

For the comparison between activated and unactivated cells, eosinophils were purified from five different volunteers. After purification, each preparation was split in half, one half was incubated with IL5 for 5 minutes and the other was left unperturbed. The ten samples were individually lysed, digested, desalted, and tagged using 10-plex Tandem Mass Tags (TMT, Thermo Scientific). The tagged samples were recombined in equal amounts and analyzed as described above except the resolution was increased for the MS2 scan to allow quantification of the reporter ions from the fragmented mass tags. The software suite COMPASS (Coon OMSSA Proteomic Analysis Software Suite) was used to analyze the data and supplemented with localization of the phosphorylation data using PhosphoRS (Thermo Scientific).

Results

An optimized sample preparation workflow coupled to high resolution mass spectrometry probed new depths of the eosinophil proteome. We identified over 100,000 unique peptides that mapped to 6,899 unique proteins. From these data we estimate the absolute abundance of 6,855 of these proteins through intensity based absolute quantification (iBAQ). Five of the 11 most abundant proteins are stored in the granules that are characteristic of eosinophils. From the IMAC-enriched samples, 5,336 sites of phosphorylation were localized. The combined protein and phosphorylation data provide unparalleled coverage of the eosinophil proteome.

To identify biological significant sites of regulatory phosphorylation we used isobaric labeling to compare the proteomes and phosphoproteomes of unactivated and IL5-activated eosinophils. The TMT reagent allowed comparison of all ten samples (five from each condition) simultaneously. Here we report the largest quantitative proteomic comparison of eosinophils to date with the quantification of 4,447 proteins and localization of 2,068 sites of phosphorylation.

Acute activation resulted in a 2-fold change in the abundance of only 5 of the 4,447 proteins (p-value of less than 0.05 from Student’s t-test with Bonferroni correction). Of identified phosphosites, however, 150 were significantly changed between the two conditions, and 20 were unique to the activated state. We are currently in the process of validating and interpreting the phosphorylation data as well as assessing significance of changes across multiple pathways. Variation among individuals will also be explored further using the current data set. These experiments should provide a solid basis for using comparative proteomics to study activation of other hematopoietic cells and variability in individual responses.