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United States Department of Agriculture

Agricultural Research Service

Title: Differential Expression Analysis of Immunosuppressed Versus Normal Neutrophil Proteome

Authors
item Lippolis, John
item Reinhardt, Timothy

Submitted to: American Society for Mass Spectrometry
Publication Type: Abstract Only
Publication Acceptance Date: February 16, 2005
Publication Date: June 6, 2005
Citation: Lippolis, J.D., Reinhardt, T.A. 2005. Differential expression analysis of immunosuppressed versus normal neutrophil proteome [abstract]. American Society for Mass Spectrometry. p. 73.

Technical Abstract: The functional activity of neutrophils from dairy cows has been shown to be drastically impaired at the time of calving. Periparturient immunosuppression is correlated with an increased incidence of mastitis. Mastitis in dairy cows results in a $2 billion annual loss for the dairy industry; thus understanding the causes of periparturient immunosuppression is of great importance. The goal of this study was to determine the differences in protein expression from normal and immunosuppressed bovine neutrophils using amine reactive isobaric tagging reagents (iTRAQ) and mass spectrometry. This information will help increase our understanding of the cellular mechanisms that cause immunosuppression. Neutrophils were isolated at, 28 days prior to calving and within 2 days of calving. Equal numbers of purified bovine neutrophils from three cows were combined and lysed. The cytosolic and membrane proteins were isolated for each sample. Equal amounts of proteins were processed using the iTRAQ kit protocol. After labeling, the samples were fractionated by strong cation exchange chromatography (SCX). SCX fractions were then analyzed by reverse phase chromatography and electro spray mass spectrometry analysis with a Waters qTOF Ultima API. Database search was performed with MASCOT 2.0 and the SwissProt protein database. All peptides used for protein identification and expression analysis had an expect score of less than 0.05 and an error tolerance of less than 20ppm. Preliminary data showed a small number of proteins were differentially expressed at calving and some of these changes may be directly involved in immunosuppression. We have identified 12 proteins that have significant expression changes at parturition out of over 125 proteins observed. Of the 12 proteins that were differentially expressed, all but one was reduced at calving. Our iTRAQ expression data showed that the expression of myeloperoxidase was down 3 fold at calving and this observation was confirmed by western blot analysis. Myeloperoxidase is an important enzyme in the generation of antimicrobial reactive oxidants. The reduction of myeloperoxidase functional activity has been strongly correlated with increased disease. In addition to myeloperoxidase, several other proteins have shown altered expression at calving and may play a role in immunosuppression. For example, our data showed that membrane bound histones were down nearly 6 fold at calving. DNA bound histones have been shown to be released by activated neutrophils as part of neutrophil extracellular traps (NETs). These NETs have been shown to have antibacterial activity. Our data suggested that NET formation was inhibited in periparturient cows, thus providing an additional potential mechanism explaining periparturient immunosuppression. Our preliminary data have shown differential regulation of several proteins known to affect neutrophil function; each could play an important part in periparturient immunosuppression. Current efforts are focused on further detection of additional differentially regulated proteins and to determine their functional role in disease resistance.

Last Modified: 7/28/2014