Single-Cell Protein Analysis

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Single-cell protein analysis
Meiye Wu and Anup K Singh
Heterogeneity of cellular systems has been widely recognized but only recently have tools become available that allow probing of genes and proteins in single cells to understand it. While the advancement in single cell genomic analysis has been greatly aided by the power of amplificationtechniques (e.g. PCR), analysis of proteins in single cells has proven to be more challenging. However, recent advances in multiparameter flow cytometry, microscopy, microfluidics and other techniques have made it possible to measure wide variety of proteins in single cells. In this review, we highlight key recent developments in analysis of proteins in a single cell (excluding imaging-based methods), anddiscuss their significance in biological research.
Address Department of Bioengineering and Biotechnology, Sandia National Laboratory, Livermore, CA 94550, United States Corresponding author: Singh, Anup K (aksingh@sandia.gov)

Current Opinion in Biotechnology 2012, 23:83–88 This review comes from a themed issue on Analytical biotechnology Edited by Wei E. Huang and Jizhong Zhou Available online19th December 2011 0958-1669/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2011.11.023

Introduction
Proteins are central to all cellular processes – including providing structure to cells, transporting molecules across cell membranes, controlling cell growth and adhesion, catalyzing biochemical processes by functioning as enzymes and regulating signaltransduction. Characterizing the quantity and activity of proteins is therefore critical for understanding molecular mechanisms of cellular processes including those involved in disease progression, cell differentiation and fate, and for targeted discovery and development of novel therapeutics, vaccines and diagnostics. Measuring DNA and RNA can provide qualitative information on gene-products(proteins) but cannot provide information on protein concentration, location, post-translational modifications (PTMs) or interactions with other proteins and hence, we need tools and assays to directly measure proteins, their modifications and interactions. Numerous analytical methods have been developed to analyze proteins such as gel electrophoresis, immunoassays, chromatography and
www.sciencedirect.commass spectrometry. However, these methods require a large number of cells for analysis, resulting in a population-averaged measurement. Cells are heterogeneous in nature and hence, population-averaged data can mask the underlying molecular mechanisms; more desirable data in many instances could be data at the level of single cells [1–5]. A well-known example is response of bacteria toantibiotics, at certain doses many die but some survive. Similarly, one of the unanswered questions in cancer therapy has been why essentially identical cells respond differently to a drug. Single-cell level measurement of proteins (and other molecules) has provided valuable insight into mechanisms that dictate heterogeneity in cellular response to drugs and other internal and external stimuli. Forexample, it was reported that dynamic response of tumor suppressor protein p53 network derived from population studies was misleading [4]. Instead of damped oscillations seen in population-averaged data, individual cells show series of undamped p53 pulses with fixed amplitude and duration, independent of the amount of g-irradiation. Similarly, real-time imaging of transcription factor RelA translocationrevealed variability in the oscillatory dynamics of RelA translocation among single cells, and that RelA translocation dynamics determined the degree and timing of downstream gene expression [3]. Usefulness of single cell measurements is obvious for stem cell research as decisions in individual cells determine their fate. For haematopoietic stem cells, studying the varying levels of Sca-1...