Neutrophils are indispensable in the innate immune defense against invading microorganisms. Neutrophils contain SVs and several subsets of granules that are essential for their function. Proteins present in neutrophil SVs and granules are synthesized during terminal granulopoiesis in the bone marrow. The heterogeneity of granules, as determined by marker proteins characteristic of each granule subset, is thought to result from differences in the biosynthetic windows of major classes of granule proteins, a process referred to as targeting by timing. Qualitative proteomic analysis of neutrophil granules, SVs, and plasma membrane has been performed before. Here, we performed subcellular fractionation on freshly isolated human neutrophils by nitrogen cavitation and density centrifugation on a four-layer Percoll gradient. Granule subsets were pooled and subjected to SDS-PAGE, and gel pieces were in-gel-digested with trypsin. The resulting peptides were analyzed using LTQ Orbitrap XL tandem MS. A total of 1292 unique proteins were identified and grouped, according to the neutrophil fraction, in which they displayed maximal expression. In addition to various known neutrophil proteins, several uncharacterized proteins were found, as well as proteins not described previously in neutrophils. To study the correlation between mRNA expression in neutrophil precursors and the localization of their cognate proteins, the distribution of 126 identified proteins was compared with their mRNA expression profiles. The neutrophil subcellular proteome profiles presented here may be used as a database in combination with the mRNA array database to predict and test the presence and localization of proteins in neutrophil granules and membranes.
Keywords: azurophil; ficolin-1; gelatinase; mass spectrometry.