single-cell-proteomics-preparation-for-mass-spectrometry-analysis

Single-Cell Proteomics Preparation for Mass Spectrometry Analysis Using Freeze-Heat Lysis and an Isobaric Carrier

Single-cell proteomics analysis requires sensitive, quantitatively accurate, widely accessible, and robust methods. To meet these requirements, the ...

Northeastern University

Increasing evidence suggests that ribosomes actively regulate protein synthesis. However, much of this evidence is indirect, leaving this layer of gene regulation largely unexplored, in part due to methodological limitations. Indeed, evidence is reviewed demonstrating that commonly used methods, such as transcriptomics, are inadequate because the variability in mRNAs coding for ribosomal proteins (RP) does not necessarily correspond to RP variability. Thus protein remodeling of ribosomes should be investigated by methods that allow direct quantification of RPs, ideally of isolated ribosomes. Such methods are reviewed, focusing on mass spectrometry and emphasizing method-specific biases and approaches to control these biases. It is argued that using multiple complementary methods can help reduce the danger of interpreting reproducible systematic biases as evidence for ribosome remodeling.

Analyzing Ribosome Remodeling in Health and Disease.

Macrophages are innate immune cells with diverse functional and molecular phenotypes. This diversity is largely unexplored at the level of single-cell proteomes because of the limitations of quantitative single-cell protein analysis.

Single-cell proteomic and transcriptomic analysis of macrophage heterogeneity using SCoPE2.

Hippo signaling is an evolutionarily conserved pathway that restricts growth and regeneration predominantly by suppressing the activity of the transcriptional coactivator Yap. Using a high-throughput phenotypic screen, we identified a potent and non-toxic activator of Yap. In vitro kinase assays show that the compound acts as an ATP-competitive inhibitor of Lats kinases-the core enzymes in Hippo signaling. The substance prevents Yap phosphorylation and induces proliferation of supporting cells in the murine inner ear, murine cardiomyocytes, and human Müller glia in retinal organoids. RNA sequencing indicates that the inhibitor reversibly activates the expression of transcriptional Yap targets: upon withdrawal, a subset of supporting-cell progeny exits the cell cycle and upregulates genes characteristic of sensory hair cells. Our results suggest that the pharmacological inhibition of Lats kinases may promote initial stages of the proliferative regeneration of hair cells, a process thought to be permanently suppressed in the adult mammalian inner ear.

Small-molecule inhibition of Lats kinases may promote Yap-dependent proliferation in postmitotic mammalian tissues.

Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying >1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Here we describe the Single Cell ProtEomics (SCoPE2) protocol, which uses an isobaric carrier to enhance peptide sequence identification. Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Preparation (mPOP), and their peptides labeled by isobaric mass tags (TMT or TMTpro) for multiplexed analysis. SCoPE2 affords a cost-effective single-cell protein quantification that can be fully automated using widely available equipment and scaled to thousands of single cells. SCoPE2 uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. The SCoPE2 workflow allows analyzing ~200 single cells per 24 h using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis.

Aleksandra A. Petelski

Department of Bioengineering

Northeastern University

Single-Cell Proteomics Preparation for Mass Spectrometry Analysis Using Freeze-Heat Lysis and an Isobaric Carrier

Aleksandra A. Petelski

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Bioengineering

Northeastern University

Single-Cell Proteomics Preparation for Mass Spectrometry Analysis Using Freeze-Heat Lysis and an Isobaric Carrier

Department of Bioengineering