title: iTRAQ is a useful method to screen for membrane-bound proteins differentially expressed in human natural killer cell types.
abstract: We are interested in the biological as well as the molecular processes involved in natural killer (NK) cell development and function. Determining the proteomic complement could be a useful tool in predicting cellular function and fate. For the first time shown here, we have utilized iTRAQ, a new method that allows identification and quantification of proteins between multiple samples, to determine the expression of membrane-bound proteins in two previously characterized human NK cell populations. One population was derived from umbilical cord blood (UCB) stem cells (CD34+38-Lin-) and the other from expanded CD3-depleted adult peripheral blood. iTRAQ was employed for multiplex peptide labeling of proteins from fractionated membranes followed by two-dimensional high-performance liquid chromatography (2D-HPLC), and tandem mass spectrometry was used to identify protein signatures. We were able to identify and quantify differences in expression levels of 400-800 proteins in a typical experiment. Ontology analysis showed the majority of the proteins to be involved in cell signaling, nucleic acid binding, or mitochondrial function. Nearly all proteins were associated with the plasma membrane, membrane-bound organelle (lysosome or mitochondria), or nucleus. We found several novel proteins highly expressed in UCB stem cell derived NK cells compared to adult NK cells including CD9, alpha-2 macroglobulin, brain abundant signaling protein (BASP1), and allograft inflammatory factor-1 (AIF-1). In addition, we were able to confirm several of our iTRAQ results by RT-PCR, Western blot, and fluorescence-activated cell-sorting (FACS) analysis. This is the first demonstration and verification using iTRAQ to screen for membrane-bound protein differences in human NK cells and represents a powerful new tool in the field of proteomics.
authors.affiliation: Department of Medicine, Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 670 CCRB, 425 East River Road, Minneapolis, MN 55455, USA. lundx072@umn.edu
authors.forename: Troy C
authors.initials: TC
authors.lastname: Lund
authors.forename: Lorraine B
authors.initials: LB
authors.lastname: Anderson
authors.forename: Valarie
authors.initials: V
authors.lastname: McCullar
authors.forename: Leeann
authors.initials: L
authors.lastname: Higgins
authors.forename: Gong H
authors.initials: GH
authors.lastname: Yun
authors.forename: Bartek
authors.initials: B
authors.lastname: Grzywacz
authors.forename: Michael R
authors.initials: MR
authors.lastname: Verneris
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Miller
mesh_terms: D000328:Adult; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000737:chemistry; D017931:DNA Primers; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry; D053804:Granzymes / Q000032:analysis; D006801:Humans; D007231:Infant, Newborn; D007694:Killer Cells, Natural / Q000737:chemistry* / Q000166:cytology / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014471:Umbilical Veins
chemical_list: D017931:DNA Primers; D008565:Membrane Proteins; D012313:RNA; D053804:Granzymes