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

Abstract

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) , IntroductionDefinition of cell form and function is derived from the transcription of specific sets of genes, followed by their translation and post-translational regulation. Cellular development alters phenotype via such changes in transcription and translation, and in this respect post-translational control of protein levels is key in many cell regulatory pathways, such as cell cycling. 1 Stem cell commitment to differentiation is a critical step in development, where proteomic changes will be observed as a cell undergoes successive commitment and developmental steps. The precise nature of the elements of gene expression that contribute to stem cell characteristics have been assessed by transcriptional profiling and the elements of a stem cell signature refined via comparative expression analyses. [2][3][4] Alternatively, the critical stem cell characteristics can be defined by analysis of the function of specific genes (in the hematopoietic system, SCL, HOX-B4, and BMI-1 5-7 ). Many key regulators are transcriptional activators or suppressors that will inevitably affect the transcriptomic profile of a cell. However, this process is known to be subject to further regulation via post-translational mechanisms. 8,9 Lin Ϫ Sca ϩ Kit ϩ (LSK ϩ ) cells and Lin Ϫ Sca ϩ Kit Ϫ (LSK Ϫ ) cells display differential abilities to reconstitute hematopoiesis, and the latter is a more mature cell type than the former, only able to support hematopoiesis in the short term. 10,11 Comparison of these 2 cell populations can help us derive knowledge of the intracellular systems that regulate the ability to maintain pluripotency and long-term self-renewal. Transcriptomic analysis, previously used to define a stem cell "signature," will not detail the true differences between stem cells and their progeny at the proteome level because of the importance of post-translational regulation of protein levels. 9 We therefore sought to establish a method for defining the proteome of stem cell populations where only limited sample is available. Furthermore, the method should not rely on transcriptome data to infer changes at the protein level. Given the limited material (approximately 1 million LSK ϩ and LSK Ϫ cells, respectively, per experiment), we developed a procedure employing isobaric tags for relative quantification (iTRAQ). This allows 4 samples to be analyzed simultaneously, giving relative quantification 12,13 on hundreds of proteins at any one time. Samples are separately proteolytically digested, and 4 isotope-coded, isobaric reagents (using differential stable isotope distribution between reporter group and balance group moieties) ...

CD34+ hematopoietic stem-progenitor cell microRNA expression and function: A circuit diagram of differentiation control

“…Taking into account the consensus that such predictions likely contains 25% false-positives (12), we estimate that 10.5% of the HSPCexpressed mRNAs are controlled by HE-miRNAs. This is in concordance with a recent study (16), which compared a limited proteome of HSPCs to various transcriptome studies (including our own study; ref. 8) and found that the expression of Ϸ15% of proteins were underestimated by their mRNA expression levels.…”

Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia

“…As HSCs are typically noncycling with less metabolic need for oxidative respiration they can reside and function adequately in a hypoxic state. Recent quantitative proteomics analysis of isolated bone marrow populations based on cell surface markers Lin Ϫ Sca ϩ Kit ϩ (LSK) indicate that these cells express high levels of glycolytic and oxidative repair proteins (37). Although this particular cell population also contains a significant amount of non-stem cell progenitors and are not as highly and exclusively enriched for HSCs as ''tip'' SP cells (24,38), it does indicate that at least some bone marrow cells are adapted to anaerobic metabolism.…”

Eight-channel iTRAQ Enables Comparison of the Activity of Six Leukemogenic Tyrosine Kinases

“…We have shown previously that oncogene-induced changes in the proteome do not always correlate to changes in the transcriptome (12). In addition changes seen in protein levels in primitive hematopoietic cells undergoing development do not correlate precisely to changes in the transcriptome (11). We therefore investigated the effects of these oncogenes on the proteome.…”

“…Investigation of protein levels in such cells is important because, in hematopoietic cells, changes in the levels of mRNA do not necessarily act as a predictor for changes in the proteome (11). The effects of the TEL/PDGFR␤ oncogene on a hematopoietic cell line proteome have been shown to be driven by altered transcription plus post-translational regulation of protein levels (12).…”