Abstract
Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ∼75-amol (∼11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or <0.2% of the total protein contained in a blastomere in the 16-cell embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level.
Highlights
From the ‡Department of Chemistry and §Department of Anatomy and Regenerative Biology, The George Washington University, Washington, DC; ¶Thomas Jefferson High School for Science and Technology, Alexandria, Virginia
We proposed that minimization of sample preparation steps that are prone to protein/peptide losses fosters higher sensitivity to enable label-free quantification (LFQ) [40] on single cells, albeit at the expense of lower sample throughput without multiplexing
We proposed that simplification of sample-handling steps prone to protein/peptide losses will enhance sensitivity to enable the quantification of single blastomeres in a label-free manner
Summary
From the ‡Department of Chemistry and §Department of Anatomy and Regenerative Biology, The George Washington University, Washington, DC; ¶Thomas Jefferson High School for Science and Technology, Alexandria, Virginia. Using microdissection to isolate single blastomeres, tandem mass tags to enable multiplexing quantification, and bottom-up proteomics, CE-ESI-HRMS was able to quantify 130 –150 different protein groups (isoforms) in common between multiple single blastomeres in the 16-cell Xenopus embryo. We proposed that minimization of sample preparation steps that are prone to protein/peptide losses fosters higher sensitivity to enable LFQ [40] on single cells, albeit at the expense of lower sample throughput without multiplexing To test this hypothesis, we adopted LFQ to our proteomic CE-ESI-HRMS platform using MaxLFQ. We adopted LFQ to our proteomic CE-ESI-HRMS platform using MaxLFQ After validation of this approach, ϳ112 different protein groups were quantified between mid-line dorsal-animal cells (D11) in the 16-cell Xenopus embryo, which are precursors of nervous tissue (brain, spinal cord, and retina) [41]. Quantification of cell-to-cell differences within the same cell type demonstrates that proteomic HRMS is sensitive enough to facilitate new types of questions in basic and translational research
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