Proteomic analysis of plasma membrane and secretory vesicles from human neutrophils
1 Buck Institute for Age Research, Novato, CA 94945, USA
2 Inflammation Program, Department of Medicine, University of Iowa and Veterans Administration Medical Center, Iowa City, IA 52240, USA
3 Howard Hughes Medical Institute, Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, Department of Molecular Physiology and Biophysics, Department of Neurology, andDepartment of Internal Medicine, University of Iowa, Iowa City, IA 52240, USA
Proteome Science 2007, 5:12 doi:10.1186/1477-5956-5-12Published: 10 August 2007
Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods.
To identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass fingerprinting) and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5-lipoxygenase-activating protein (FLAP) and dysferlin were further validated by immunoblot analysis.
Our data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli.