Toward the proteome of the human peripheral blood eosinophil

Abstract

Eosinophils are granular leukocytes that have significant roles in many inflammatory and immunoregulatory responses, especially asthma and allergic diseases. We have undertaken a fairly comprehensive proteomic analysis of purified peripheral blood eosinophils from normal human donors primarily employing 2-dimensional gel electrophoresis with protein spot identification by matrix-assisted laser desorption/ionization mass spectrometry. Protein subfractionation methods employed included isoelectric focusing (Zoom® Fractionator) and subcellular fractionation using differential protein solubilization. We have identified 3,141 proteins which had Mascot expectation scores of 10−3 or less. Of these 426 were unique and non-redundant of which 231 were novel proteins not previously reported to occur in eosinophils. Ingenuity Pathway Analysis showed that some 70% of the non-redundant proteins could be subdivided into categories that are clearly related to currently known eosinophil biological activities. Cytoskeletal and associated proteins predominated among the proteins identified. Extensive protein posttranslational modifications were evident, many of which have not been previously reported that reflected the dynamic character of the eosinophil. This dataset of eosinophilic proteins will prove valuable in comparative studies of disease versus normal states and for studies of gender differences and polymorphic variation among individuals.

IL-5 Induces Suspended Eosinophils to Undergo Unique Global Reorganization Associated with Priming

“…Loss of lamins from the nuclear envelope impairs nuclear positioning in fibroblasts and skeletal muscle (37,38). Eosinophils express lamin B1 (39,40), and, in a pilot experiment, we localized lamin B1 in the nuclear envelope ( Figure E15). Thus, the difference between the nuclear envelopes of neutrophils and eosinophils may also contribute to the different locations of their nuclei after activation.…”

Altered Eosinophil Proteome in a Patient with Hypereosinophilia from Acute Fascioliasis

“…Granulocytes were isolated by gradient centrifugation and erythrocytes lysed with hypotonic saline as described previously (10). Granulocytes were incubated with anti-CD16 immunomagnetic beads and loaded onto a VarioMACS (Miltenyi Biotec, Auburn CA) column, and a magnetic field was used to separate neutrophils from eosinophils.…”

“…Granulocytes were incubated with anti-CD16 immunomagnetic beads and loaded onto a VarioMACS (Miltenyi Biotec, Auburn CA) column, and a magnetic field was used to separate neutrophils from eosinophils. The isolated eosinophils (Ͼ97% pure by Giemsa staining) were lysed in DeStreak rehydration buffer, frozen (Ϫ70 C), and transported on dry ice to the University of Texas Medical Branch, Galveston, TX, where the whole-cell lysate was resolved via 2-dimensional electrophoresis (2-DE) as described previously (10). Gels were stained with Sypro Ruby protein stain (Bio-Rad, Hercules CA) and imaged, and the normalized volumes of protein spots compared to a baseline eosinophil proteome map from control donors using Nonlinear SameSpots software (Nonlinear Dynamics, Durham NC).…”

“…Gels were stained with Sypro Ruby protein stain (Bio-Rad, Hercules CA) and imaged, and the normalized volumes of protein spots compared to a baseline eosinophil proteome map from control donors using Nonlinear SameSpots software (Nonlinear Dynamics, Durham NC). Proteins differentially expressed by Ն2-fold were excised and analyzed by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF-TOF MS) (Applied Biosystems 4800) (10). Applied Biosystems GPS ExplorerTM (version 3.6) software was used along with MASCOT (Matrix Science, London, United Kingdom) to search protein databases using both MS and tandem MS (MS-MS) spectral data for protein identification.…”

“…1) (10). Of the 60 protein spots that were differentially expressed, some 44 were subjected to MALDI-TOF-TOF MS, and their identifications are given in Table 1.…”