Overall experimental design. (A) Flow diagram showing the steps involved in determining relative quantities of proteins between LSK⁺ cells and LSK^– cells and the comparison of these data to transcriptome data obtained on the same samples. (B) UV absorbance spectrum (214 nm) showing fractionation of iTRAQ-labeled peptides by strong cation exchange chromatography. The trace shows the elution profile of the cell lysates. The gradient is illustrated by the dotted line, and the range during which 1-minute fractions were collected is marked with an arrow. (C) Separation of a single SCX fraction (fraction 22) on a reverse phase gradient on-line to a mass spectrometer. The trace shows the total ion chromatograph (TIC) indicated as intensity on the y-axis, showing the elution profile of peptides from the reverse phase high-performance liquid chromatography (HPLC) column into the mass spectrometer. (D) Tandem mass spectrum showing the sequence of a peptide of mass: charge 583.37 eluting from the reverse phase column at 53.6 minutes. The labeled y-ion series allows calculation of the peptide sequence (SFVLNLGJ, where J denotes a lysine with an iTRAQ-labeled side-chain); the iTRAQ reporter ions (inset) give relative quantification data for this peptide.