The potential for proteomic definition of stem cell populations Richard D Unwin, Simon J Gaskell, Caroline A Evans, Anthony D Whetton  Experimental Hematology  Volume 31, Issue 12, Pages 1147-1159 (December 2003) DOI: 10.1016/j.exphem.2003.08.012

Figure 1 Typical workflow for using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to identify differentially expressed proteins in two different samples. The key aspects are visualization to obtain relative quantification of material from two samples, the ability to select a protein spot from those surrounding it, and the use of mass spectrometry (MS) to identify the protein in question. In practice, gel-based technologies limit the amount of protein recovered, silver stain more so than fluorescent stains in our experience, thereby affecting the ability to identify low-abundance proteins. Experimental Hematology 2003 31, 1147-1159DOI: (10.1016/j.exphem.2003.08.012)

Figure 2 Two-dimensional gel containing 2.5×105 Lin−Sca+Kit− hematopoietic cell protein lysate prelabeled with a saturation Cy5 dye and electrophoresed over an 24-cm pH 3–10 nonlinear gradient in the first dimension and SDS-PAGE resolving gel in the second dimension. About 900 spots can be seen on this gel. Protein from a similar number of Lin−Sca+Kit+ cells was prelabeled with Cy3 dye and electrophoresed on the same gel (not shown), allowing intragel comparison of the major proteins expressed by these two stem cell populations. Such proteins can be excised and identified using mass spectrometry. Experimental Hematology 2003 31, 1147-1159DOI: (10.1016/j.exphem.2003.08.012)

Figure 3 Schematic representation of the workflow for a comparative proteomics experiment using stable isotope labeling and mass spectrometry. The derivatization chemistry used to label proteins from the two experimental samples is key to this process. The reaction must go to completion, must enable simple identification of peptide pairs in a mass spectrum (thereby giving relative quantitation), and must not adversely affect either protein digestion or the behavior of the labeled peptides (either during separation by liquid chromatography or within the mass spectrometer). Some chemistries, e.g., isotope-coded affinity tag (ICAT; see text for discussion), contain a biotin moiety to enable enrichment/purification of labeled peptides. Isotope coding of proteins by growing samples in culture with labeled amino acids is described in the text, as is use of post-lysis derivatization chemistries with isotope coding reagents. D=deuterium; 15N, 14N=isotopes of nitrogen used to generate isotope-coded amino acids for differential protein labeling in culture. Experimental Hematology 2003 31, 1147-1159DOI: (10.1016/j.exphem.2003.08.012)