We are interested in the biological as well as molecular processes involved in natural killer cell differentiation and function based on the growing interest in these cells for cancer therapy and allogeneic transplantation. Determining the proteomic complement is a useful tool in predicting cellular function and fate. iTRAQ is a new tandem mass-spec method which not only allows identification of proteins in the picomole to femtomole range, but also allows one to quantify differences in specific protein amount between two samples. Using this method one can potentially get reliable proteomic information from cells and also compare expression differences between cell populations. For the first time shown here, we have applied the new method of iTRAQ to identify and quantify the expression of membrane localized proteins in two previously characterized distinct but related NK cells populations. One population was derived in vitro from umbilical cord blood stem cells (CD34+Lin−CD38−) differentiated with murine stroma and human cytokines (IL-3, Flt3L, SCF, IL-7. IL-15) and the other from NK cells expanded from CD3-depleted adult peripheral blood and IL-2, both resulting in cultured populations of >90% NK cells. iTRAQ was employed for multiplex peptide labeling followed by 1- and 2-dimensional high performance liquid chromatography (1D- and 2D-HPLC) and then tandem mass spectroscopy was used to analyze the proteomic contingent of membrane associated proteins. We were able to identify well as quantify differences in expression levels of 35–133 proteins in a typical 1D-HPLC experiment. This increased to >400 proteins when 2D-HPLC was used. Over 75% of the proteins were identified with more than 2 peptide fragments. Gene ontogeny analysis showed the majority of proteins to be involved in cell signaling, nucleic acid binding, or cell adhesion. Nearly all proteins were associated with the plasma membrane, membrane bound organelle (lysosome or peroxisome), or nucleus. Several unique proteins were found which previously have not been known to be expressed by NK cells: BASP1, nmb, and AIF-1. More important, we were able to confirm several of our iTRAQ results by RT-PCR, Western blot, and FACS analysis. This is the first demonstration, and verification of using iTRAQ to determine the “membrane proteome” in NK cells and represents a valid and powerful new tool in the science of proteomics. These novel differences between NK cells and their precursors will be studied further to understand NK cell interactions and novel mechanisms which govern NK cell development.

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