Fig. 3.

MS/MS spectra of peptides with corresponding iTRAQ^TM reporter intensities identified in CD56⁺ NK cell subsets. Sorted NK cells were lysed, and trypsin-digested peptides were tagged with iTRAQ-label 115 (for CD56^bright NK cell subset) and 117 (for CD56^dim NK cell subset), or with 114 (for CD57⁺ NK cell subset) and 116 (for CD57⁻ NK cell subset). Subset peptides (CD56^dimversus CD56^bright, CD57⁺versus CD57⁻) were combined in 1:1 ratio and sequenced via nanoLC-MS/MS. Peptide fragmentation detected b (red)- and y (blue)-ions. Peptide sequences were derived from these ions through defined amino acid masses. Through Mascot-based database searches in UniProt/Swiss-Prot, peptide sequences were assigned to the proteins CD56 (A) and Perforin (B). Quality of this assignment depends on high peptide Mascot Scores. iTRAQ reporters occur in low mass regions of the spectrum (green). By comparing normalized reporter intensities (115-ref against 117 and 114 against 116-ref), peptide abundances were quantified and log₂-regulation factors (RFs) were generated. The RFs of all identified peptides of one protein represent the corresponding protein regulation factor.