iTRAQ Is a Useful Method To Screen for Membrane-Bound Proteins Differentially Expressed in Human Natural Killer Cell Types

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.
Keywords: Natural killer cell · tandem mass spectrometry · iTRAQ · umbilical cord blood · liquid chromatography
· membrane-bound