Camille Lombard-Banek (Presenter)
The George Washington University
Bio: I am currently in my 3rd year of the PhD program at the George Washington University. My main research interest is in developing new tools for the analysis of volume-limited biological samples, more specifically for the proteomic analysis of single cells.
Authorship: Camille Lombard-Banek, Sally A. Moody and Peter Nemes
(1) Department of Chemistry and (2) Department of Anatomy & Regenerative Biology, The George Washington University
| Short Abstract Characterization of the proteomic machinery underlying cell differentiation promises to elevate our understanding of the normal and impaired development of the vertebrate embryo. However, this requires specialized, highly sensitive tools, particularly based on mass spectrometry, to measure single cells. Here, we utilize a custom-built capillary electrophoresis microelectrospray mass spectrometry technique to study proteomic differences between selected blastomeres that were extracted from the 16-cell normal frog embryo. This technology enabled the reproducible quantification of ~150 nonredundant proteins, which allowed us to uncover translational differences between multiple blastomere types. These results set the stage for translational studies where the hindrance of developmental pathways is suspected to lead to inborn defects in the embryo. |
Long Abstract
Introduction. Characterization of biomolecules in single cells raises new opportunities for better understanding developmental mechanisms and identifying biomarkers for developmental disorders. In turn, such information promises to help the development of targeted therapeutics, to remediate impairments affecting the vertebrate system, ranging from the degeneration of the muscle to nervous system. Proteins are of particular interest as biomarkers because they provide an informative read on the cell’s state and phenotype, providing a plethora of information concerning the cell homeostasis. However, proteomic measurements on single cells require new technologies that are capable of enabling the identification and quantitation of a large number of proteins, while preferably operating in an untargeted manner. Here, we describe one such technology that we have developed for single cells: capillary electrophoresis microflow electrospray ionization mass spectrometry (CE-µESI-HRMS). We demonstrate that this platform allows for the analysis of nanoliters (nanograms) of proteins, making it possible to determine the translational state of single embryonic cells (blastomeres) in the 16-cell South African clawed frog (Xenopus laevis) embryos.
Methods. Single identified Xenopus blastomeres were manually isolated from the 16-cell embryo using surgical forceps using established protocols. The cells were lysed in 10 µL 1% sodium dodecyl sulfate, facilitated by sonication for ~5 min. The resulting protein extract was reduced by dithiotreitol, alkylated using iodoacetamide, and precipitated in cold acetone (–20°C). The proteins were pelleted via centrifugation, washed with chilled acetone, and reconstituted in 50 mM ammonium bicarbonate before tryptic digestion. The digests were lyophilized and resuspended in 5 µL 50% acetonitrile containing 0.05% acetic acid. A ~20-nL fraction of this single-cell digest was measured by a custom-built microcapillary electrophoresis electrospray ionization (µCE-ESI) platform that was coupled to a qQ-Q-time-of-flight (Impact HD, Bruker) or an q-orbitrap-linear ion trap (Fusion, Thermo) tandem mass spectrometer. Proteins were identified with <1% false discovery rate (FDR) using Proteome Discoverer executing SEQUEST search engine against the NCBInr database for Xenopus or a proteomic database that was recently developed based on experimentally determined mRNA expression in the Xenopus embryo.
Results. We first characterized the analytical figures of merits for our custom-built CE-µESI-HRMS platform using peptide standards. The lower limit of detection was established at ~25 amol for angiotensin II and leucine encephalin standard peptides. Using whole-embryo digests, the quantitative reproducibility was determined at ~15% standard error of the mean for both technical (sample extract measured multiple times) and biological replicates (different embryos analyzed). We then iteratively refined each step of the bottom-up proteomic workflow using whole embryos to progressively increase the identifiable the number of proteins from ~10 to ~500 protein groups. This level of identification of the proteome provided leverage to the applicability of our system for measuring single blastomeres.
To determine translational differences between blastomeres, we chose the D11, V11 and V21 cells in the 16-celll embryo; these cells have different positions in the embryo and give rise to different types of tissues later during development. The single blastomeres were identified based in their pigmentation and manually dissected following protocols that we have also established. The cells were lysed, and the resulting extracts (~10 µg of total protein/cell) were digested using trypsin. The digests were lyophilized and reconstituted in 5 µL of a solution containing 50% acetonitrile in water with 0.05% formic acid. With low sample consumption, ~20 ng/measurement, possible by the CE-µESI-MS platform, we were able measure each blastomere in multiple technical replicates, thus enhancing the reproducibility of measurements and also the number of protein identifications. By measuring each cell in technical duplicates and biological triplicates, we identified ~890 proteins in each cell type on average, amounting to a total of 1,709 different protein groups between all replicates. Exponentially modified protein abundance indices (emPAI) that were calculated based on the mass spectrometric data suggested that the concentration range of these proteins spanned ~4-to-5 orders of magnitude. Statistical analysis using analysis of variance (ANOVA) between the blastomeres found ~300 proteins to be differentially expressed between the D11, V11, and V21 cell types.
To confirm these findings using an orthogonal approach, we extended our technology and single-cell analysis protocols to quantitation using tandem mass tags (TMTs). As a result, we were able to quantify ~150 nonredundant protein groups across all the blastomeres. Based on these quantitative data, several of these proteins were found to exhibit significantly different levels depending on cell type; these differences were statistically (t-test, p < 0.05) and biologically significant (fold change > 1.5). The differences contained 11 protein groups between D11–V11, 35 protein groups between D11–V21, and 22 protein groups between the V11–V21 cell types. Several of these proteins are known to be involved in blastomere fate determination, corroborating our measurement data. For example, the translation initiation factor Eif5a was accumulated in D11 blastomeres. In agreement, this protein is involved in brain development, and brain is a stereotypical fate of the D11 blastomeres. Strikingly, our results uncovered proteomic differences not only between animal and vegetal hemispheres of the embryo but also between blastomeres that occupy the dorsal vs. ventral sides of the embryo. This finding was surprising because the transcriptome reveals cell-to-cell differences only along the animal-vegetal axis, but not the dorsal-ventral in the 16-cell embryo.
Conclusions. Single-cell mass spectrometry allowed us to uncover translational differences between blastomeres that were previously unknown, providing new data for cell and developmental biology. We expect this technology to be widely applicable for other developmental models as well as smaller embryonic cells from Xenopus or other models.
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