High sensitivity top–down proteomics captures single muscle cell heterogeneity in large proteoforms

Proceedings of the National Academy of Sciences, Journal Year: 2023, Volume and Issue: 120(19)

Published: May 1, 2023

Single-cell proteomics has emerged as a powerful method to characterize cellular phenotypic heterogeneity and the cell-specific functional networks underlying biological processes. However, significant challenges remain in single-cell for analysis of proteoforms arising from genetic mutations, alternative splicing, post-translational modifications. Herein, we have developed highly sensitive functionally integrated top–down comprehensive single cells. We applied this muscle fibers (SMFs) resolve their heterogeneous proteomic properties at level. Notably, detected large (>200 kDa) SMFs. Using SMFs obtained three distinct muscles, found fiber-to-fiber among sarcomeric which can be related heterogeneity. Importantly, multiple isoforms myosin heavy chain (~223 kDa), motor protein that drives contraction, with high reproducibility enable classification individual fiber types. This study reveals cell establishes direct relationship between types, highlighting potential uncovering molecular underpinnings cell-to-cell variation complex systems.

Language: Английский

---

Proximity Labeling in Plants

Annual Review of Plant Biology, Journal Year: 2023, Volume and Issue: 74(1), P. 285 - 312

Published: March 1, 2023

Proteins are workhorses in the cell; they form stable and more often dynamic, transient protein-protein interactions, assemblies, networks have an intimate interplay with DNA RNA. These network interactions underlie fundamental biological processes play essential roles cellular function. The proximity-dependent biotinylation labeling approach combined mass spectrometry (PL-MS) has recently emerged as a powerful technique to dissect complex at molecular level. In PL-MS, by fusing genetically encoded proximity-labeling (PL) enzyme protein or localization signal peptide, is targeted of interest organelle, allowing proximity proteins within zoom radius. biotinylated can then be captured streptavidin beads identified quantified spectrometry. Recently engineered PL enzymes such TurboID much-improved enzymatic activity, enabling spatiotemporal mapping dramatically increased signal-to-noise ratio. PL-MS revolutionized way we perform proteomics overcoming several hurdles imposed traditional technology, biochemical fractionation affinity purification this review, focus on biotin ligase-based applications that been, likely be, adopted plant field. We discuss experimental designs review different choices for ligases, enrichment, quantification strategies. Lastly, validation future perspectives.

Language: Английский

---

Evaluating the capabilities of the Astral mass analyzer for single-cell proteomics

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: June 8, 2023

Abstract The complexity of human physiology arises from well-orchestrated interactions between trillions single cells in the body. While single-cell RNA sequencing (scRNA-seq) has enhanced our understanding cell diversity, gene expression alone does not fully characterize phenotypes. Additional molecular dimensions, such as proteins, are needed to define cellular states accurately. Mass spectrometry (MS)-based proteomics emerged a powerful tool for comprehensive protein analysis, including applications. However, challenges remain terms throughput and proteomic depth, order maximize biological impact by Spectrometry (scp-MS) workflows. This study leverages novel high-resolution, accurate mass (HRAM) instrument platform, consisting both an Orbitrap innovative HRAM Asymmetric Track Lossless (Astral) analyzer. Astral analyzer offers high sensitivity resolution through lossless ion transfer unique flight track design. We evaluate performance Thermo Scientific MS using Data-Independent Acquisition (DIA) assess proteome depth quantitative precision ultra-low input samples. Optimal DIA method parameters identified, we demonstrate ability cycle dynamics Human Embryonic Kidney (HEK293) cells, cancer heterogeneity primary Acute Myeloid Leukemia (AML) culture model.

Language: Английский

---

HyperSCP: Combining Isotopic and Isobaric Labeling for Higher Throughput Single-Cell Proteomics

Analytical Chemistry, Journal Year: 2023, Volume and Issue: 95(20), P. 8020 - 8027

Published: May 11, 2023

Recent developments in mass spectrometry-based single-cell proteomics (SCP) have resulted dramatically improved sensitivity, yet the relatively low measurement throughput remains a limitation. Isobaric and isotopic labeling methods been separately applied to SCP increase through multiplexing. Here we combined both forms of achieve multiplicative scaling for higher throughput. Two-plex stable isotope amino acids cell culture (SILAC) isobaric tandem tag (TMT) enabled up 28 single cells be analyzed liquid chromatography–mass spectrometry (LC–MS) analysis, addition carrier, reference, negative control channels. A custom nested nanowell chip was used nanoliter sample processing minimize losses. Using 145-min total LC–MS cycle time, ∼280 were per day. This could increased ∼700 samples day with high-duty-cycle multicolumn LC system producing same active gradient. The efficiency achievable proteome coverage characterized multiple analysis conditions.

Language: Английский

---

Single‐cell proteomics differentiates Arabidopsis root cell types

New Phytologist, Journal Year: 2024, Volume and Issue: 244(5), P. 1750 - 1759

Published: June 24, 2024

Single-cell proteomics (SCP) is an emerging approach to resolve cellular heterogeneity within complex tissues of multi-cellular organisms. Here, we demonstrate the feasibility SCP on plant samples using model Arabidopsis thaliana. Specifically, focused examining isolated single cells from cortex and endodermis, which are two adjacent root cell types derived a common stem lineage. From 756 cells, identified 3763 proteins 1118 proteins/cell. Ultimately, focus 3217 quantified following stringent filtering. Of these, 596 whose expression enriched in either or endodermis able differentiate these closely related types. Collectivity, this study demonstrates that can neighboring with distinct functions, thereby facilitating identification biomarkers candidate enable functional genomics.

Language: Английский

---