This proposal is a competitive renewal. In the 3.5 years since we received funding, we have published 29 papers acknowledging support from this grant; another five papers are under review. These publications address issues in diagonal capillary electrophoresis, including reactor design and improved capillary zone electrophoresis separations. We and others have demonstrated that capillary zone electrophoresis consistently provides more protein and peptide identifications than high performance liquid chromatography for mass-limited samples. We will take advantage of our experience to develop and evaluate technology for the bottom-up proteomic analysis of single blastomeres.
Our first aim will develop on-column sample preconcentration technology, coupled with capillary zone electrophoresis peptide separation and an electrokinetically-pumped sheath flow nanoelectrospray interface with tandem mass spectrometry detection.
Our second aim will develop on-column cell lysis, reduction and alkylation, digestion, and preconcentration in an integrated device, which will be evaluated using blastomeres along the D1 lineage.
Our third aim provides bioinformatic support and ensures wide distribution of our results. This project also builds off of a recent publication from this team, which reported the first quantitative proteomic analysis of a developing embryo (Sci Rep. 2014; 4: 4365. PMID: 24626130). We studied single Xenopus laevis embryos at six stages of development, including stage 1 embryos. These stage 1 embryos have not divided, and constitute a single cell; we quantified the expression of 4,000 proteins from these single cells. We will extend our Sci Rep publication by quantitatively monitoring protein expression in single blastomeres from stage 2 of development through stage 20. These cells contain from 50-g to 50-pg of protein, and form a natural progression of cells with progressively smaller protein content, ultimately ending at the size of a typical mammalian somatic cell. This progression of cell sizes is ideal for the development and evaluation of technology for single cell analysis. We will focus on the analysis of single cells isolated from the lineage of the blastomere D1, which ultimately form portions of the adult retina, spinal cord, and brain. Proteomic analysis of single blastomeres isolated from this lineage will provide insight into the assembly of the organism's nervous system.

Public Health Relevance

Proteomics provides a detailed enumeration of the identity and abundance of proteins in complex samples. Those analyses typically require micrograms of sample. This proposal will develop and evaluate technology for quantitative proteomics of much smaller samples, ranging in protein content from 50-g to 50-pg. The technology will be applied to important issues in developmental biology, but the technology will also find application whenever dealing with mass-limited samples, such as circulating tumor cells, fine needle aspirant biopsies, or laser-capture microdissected tissues.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
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Krepkiy, Dmitriy
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University of Notre Dame
Schools of Arts and Sciences
Notre Dame
United States
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Peuchen, Elizabeth H; Cox, Olivia F; Sun, Liangliang et al. (2017) Phosphorylation Dynamics Dominate the Regulated Proteome during Early Xenopus Development. Sci Rep 7:15647
Flaherty, Ryan J; Sarver, Scott A; Sun, Liangliang et al. (2017) A High Voltage Power Supply That Mitigates Current Reversals in Capillary Zone Electrophoresis-Electrospray Mass Spectrometry. J Am Soc Mass Spectrom 28:247-252
Peuchen, Elizabeth H; Zhu, Guije; Sun, Liangliang et al. (2017) Evaluation of a commercial electro-kinetically pumped sheath-flow nanospray interface coupled to an automated capillary zone electrophoresis system. Anal Bioanal Chem 409:1789-1795
Schmudlach, Andrew; Felton, Jeremy; Kennedy, Robert T et al. (2017) Bottom-up proteomics analysis of the secretome of murine islets of Langerhans in elevated glucose levels. Analyst 142:284-291
Sarver, Scott A; Schiavone, Nicole M; Arceo, Jennifer et al. (2017) Capillary electrophoresis coupled to negative mode electrospray ionization-mass spectrometry using an electrokinetically-pumped nanospray interface with primary amines grafted to the interior of a glass emitter. Talanta 165:522-525
Krokhin, Oleg V; Anderson, Geoffrey; Spicer, Vic et al. (2017) Predicting Electrophoretic Mobility of Tryptic Peptides for High-Throughput CZE-MS Analysis. Anal Chem 89:2000-2008
Zhang, Zhenbin; Peuchen, Elizabeth H; Dovichi, Norman J (2017) Surface-Confined Aqueous Reversible Addition-Fragmentation Chain Transfer (SCARAFT) Polymerization Method for Preparation of Coated Capillary Leads to over 10 000 Peptides Identified from 25 ng HeLa Digest by Using Capillary Zone Electrophoresis-Tandem Ma Anal Chem 89:6774-6780
Boley, Danielle A; Zhang, Zhenbin; Dovichi, Norman J (2017) Multisegment injections improve peptide identification rates in capillary zone electrophoresis-based bottom-up proteomics. J Chromatogr A 1523:123-126
Sun, Liangliang; Dubiak, Kyle M; Peuchen, Elizabeth H et al. (2016) Single Cell Proteomics Using Frog (Xenopus laevis) Blastomeres Isolated from Early Stage Embryos, Which Form a Geometric Progression in Protein Content. Anal Chem 88:6653-7
Zhu, Guijie; Sun, Liangliang; Dovichi, Norman J (2016) Thermally-initiated free radical polymerization for reproducible production of stable linear polyacrylamide coated capillaries, and their application to proteomic analysis using capillary zone electrophoresis-mass spectrometry. Talanta 146:839-43

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