Single Cell Proteomics by Data-Independent Acquisition To Study Embryonic Asymmetry in Xenopus laevis

Abstract

Techniques that allow single cell analysis are gaining widespread attention and most of these studies utilize genomics-based approaches. While nanofluidic technologies have enabled mass spectrometric analysis of single cells, these measurements have been limited to metabolomics and lipidomic studies. Single cell proteomics has the potential to improve our understanding of intercellular heterogeneity. However, this approach has faced challenges including limited sample availability, as well as a requirement of highly sensitive methods for sample collection, clean-up and detection. We present a technique to overcome these limitations by combining a micropipette (pulled glass capillary) based sample collection strategy with offline sample preparation and nanoLC-MS/MS to analyze proteins through a bottom-up proteomic strategy. This study explores two types of proteomics data acquisition strategy namely data-dependent (DDA) and dataindependent acquisition (DIA) strategy. Results from the study indicate DIA to be more sensitive enabling analysis of >1600 proteins from ~130 μm Xenopus laevis embryonic cells containing <6 nL of cytoplasm. The method was found to be robust in obtaining reproducible protein *

Quantitative single-cell proteomics as a tool to characterize cellular hierarchies

“…Others have since taken a similar multiplexed approach using a carrier channel in combination with a cutting-edge sample preparation technique known as NanoPOTS, and demonstrated very promising results when sample loss is decreased to a minimum [27][28][29] . Although label-free approaches have also shown promising results [30][31][32][33][34] , their throughput currently lags behind the multiplexed approach. Comprehensive evaluations of multiplexed scMS using an isobaric carrier 29,35,36 have further demonstrated the feasibility of the approach, concluded on the tradeoffs of increasing the level of signal boosting with the isobaric carrier, and indicated the importance of estimating the reliability of protein quantification when implementing the method.…”

Patch-Clamp Proteomics of Single Neuronal Somas in Tissue Using Electrophysiology and Subcellular Capillary Electrophoresis Mass Spectrometry

“…Standard bottom-up proteomics requires extraction and digestion of the proteins, often also calling for chemical reduction/alkylation to deepen the coverage of the detectable proteome. These starting protein amounts, however, were ~1-10 million times smaller than traditionally processed in MSbased bottom-up proteomics, ~1-100 thousand times less than handled from single dissected cells, 29,32,49 and ~100-times smaller than recently collected from individual cells using capillary microsampling 33,34,50 . As a simplified alternative to automated droplet processing [18][19][20][21] , we opted to carry out the processing steps in Lo-Bind vials, common consumables in laboratories (see Methods).…”

Single‐Cell Microgels for Diagnostics and Therapeutics

“…[309] To make single-cell proteomics a mainstream reality, highly sensitive methods for sample collection, clean-up, and detection are required. [310] Even with these sizeable obstacles, scientists are still optimistic about implementing single-cell proteomics to directly analyze full protein profiles. Unlike transcriptomic studies, no amplification of cellular protein is needed due to its natural, high abundance, even in a single cell.…”