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12 | SEPTEMBER 1, 2016 | GENengnews.com | Genetic Engineering & Biotechnology News Dynamic Shifts in Protein Profiling demonstrating a proteomic outlook. It is part of a shift from protein identification to protein quantification, the measure- ment of protein expression levels. Moreover, protein profil- ing now encompasses protein structure-function relation- ships and protein-protein interactions. Protein profiles may reflect not only the fluctuations in protein levels, but also the shifts between active and inactive protein forms. As always, protein profiles are about comparisons—dis- eased vs. healthy, treated vs. untreated, experimental vs. control—but now the comparisons have more depth. They are like census-based analyses that substantiate increasingly fine demographic distinctions. Protein profiling is becoming more refined through the use of advanced experimental and analytical approaches, several of which are sum- marized here. Reconstructing Differentiation Trajectories "A key limitation at the moment is that we are constrained by the number of proteins that we can measure by flow cy- tometry in a single cell," says Berthold Göttgens, Ph.D., pro- fessor of molecular hematology at the University of Cam- bridge. Current flow cytometry approaches allow only up to 20 to 30 protein species to be simultaneously profiled in a cell, and the technology typically provides a snapshot of cellular information. Dr. Göttgens' laboratory uses both experimental and computational approaches to study the transcriptional con- trol of normal and leukemic blood stem/progenitor cells. Transcriptional control, Dr. Göttgens reasons, needs to be assessed at the level of single cells, not the bulk of cells. Fortunately, cells of different types—which are, presumably, transcrip- tionally distinct—may be isolated from the bulk ac- c o r d i n g to their surface maker proteins. In stem cell biology, population-level pro- filing provides only population averages and is uninforma- tive about the biology of single cells. The importance of cap- turing single-cell processes, increasingly recognized in many areas of life sciences, is particularly critical for stem cell re- search, where progression between distinct cellular states is fundamental for understanding the biology and for develop- ing therapeutic interventions. In a recent study stemming from practical challenges caused by molecular and functional heterogeneities of mu- rine hematopoietic stem cells, Dr. Göttgens and colleagues combined single-cell gene-expression analyses, flow cytomet- ric sorting, and functional assays to better understand the gene-expression program at the single-cell level. "Establish- ing a connection between surface marker protein profiles and a whole transcriptome helps use the surface markers to purify viable cells," explains Dr. Göttgens. "Ultimately, this approach can be used for applications with potential thera- peutic benefits." By taking advantage of recent molecular-profiling tech- nologies, Dr. Göttgens and colleagues interrogated early he- matopoietic stem cell differentiation at the single-cell level in mouse hematopoietic progenitor stem cells. Surface-based cell sorting was used to retrospectively assign cells to one of twelve different phenotypes. "From these same cells, we also recorded mRNA expres- sion for the entire transcriptome," details Dr. Göttgens. This helped link single-cell gene-expression profiles with single- cell function. An online repository that incorporated the data provided a resource to visualize lineage-specific tran- scriptional programs and helped generate an atlas of the early hematopoietic stem cell differentiation at the single-cell resolution. "Linking molecular profiles with surface marker profiles enabled us to detect protein expression levels without the need to lyse the cells," explains Dr. Göttgens. Measuring sur- face protein markers and gene expression in the same single cells, and connecting the two, allowed investigators to use the surface markers to specifically purify distinct populations of cells. DRUG DISCOVERY Measuring surface protein markers and gene expression in the same cells allowed investigators to use the markers to purify distinct populations of cells. See Protein Profiling on page 14 Continued from page 1 A multi-protease strategy on the HeLa proteome to improve protein sequence coverage, and to target regions of proteins that do not generate useful tryptic peptides.