Quantitative proteomics reveals posttranslational control as a regulatory factor in primary hematopoietic stem cells

• Published in: Blood Vol. 107; no. 12; pp. 4687 - 4694
• Main Authors: Unwin, Richard D., Smith, Duncan L., Blinco, David, Wilson, Claire L., Miller, Crispin J., Evans, Caroline A., Jaworska, Ewa, Baldwin, Stephen A., Barnes, Kay, Pierce, Andrew, Spooncer, Elaine, Whetton, Anthony D.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.06.2006
• Subjects: Animals; Cell Hypoxia; Chromatography, Liquid; Hematopoietic Stem Cells - physiology; Mass Spectrometry; Mice; Oxidation-Reduction; Protein Processing, Post-Translational - physiology; Proteome - genetics; Proteome - metabolism; Proteomics
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2005-12-4995

The proteome is determined by rates of transcription, translation, and protein turnover. Definition of stem cell populations therefore requires a stem cell proteome signature. However, the limit to the number of primary cells available has restricted extensive proteomic analysis. We present a mass spectrometric method using an isobaric covalent modification of peptides for relative quantification (iTRAQ), which was employed to compare the proteomes of approximately 1 million long-term reconstituting hematopoietic stem cells (Lin–Sca+Kit+; LSK+) and non–long-term reconstituting progenitor cells (Lin–Sca+Kit–; LSK–), respectively. Extensive 2-dimensional liquid chromatography (LC) peptide separation prior to mass spectrometry (MS) enabled enhanced proteome coverage with relative quantification of 948 proteins. Of the 145 changes in the proteome, 54% were not seen in the transcriptome. Hypoxia-related changes in proteins controlling metabolism and oxidative protection were observed, indicating that LSK+ cells are adapted for anaerobic environments. This approach can define proteomic changes in primary samples, thereby characterizing the molecular signature of stem cells and their progeny.