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

Proceedings of the National Academy of Sciences · 2023 · 66 citations

• Computer Science
• Computer Science
• Engineering

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

GH Action in Prostate Cancer Cells Promotes Proliferation, Limits Apoptosis, and Regulates Cancer-related Gene Expression

Endocrinology · 2022 · 16 citations

• Endocrinology
• Internal medicine
• Biology

Previous studies investigating the effects of blocking the growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis in prostate cancer found no effects of the growth hormone receptor (GHR) antagonist, pegvisomant, on the growth of grafted human prostate cancer cells in vivo. However, human GHR is not activated by mouse GH, so direct actions of GH on prostate cancer cells were not evaluated in this context. The present study addresses the species specificity of GH-GHR activity by investigating GH actions in prostate cancer cell lines derived from a mouse Pten-deletion model. In vitro cell growth was stimulated by GH and reduced by pegvisomant. These in vitro GH effects were mediated at least in part by the activation of JAK2 and STAT5. When Pten-mutant cells were grown as xenografts in mice, pegvisomant treatment dramatically reduced xenograft size, and this was accompanied by decreased proliferation and increased apoptosis. RNA sequencing of xenografts identified 1765 genes upregulated and 953 genes downregulated in response to pegvisomant, including many genes previously implicated as cancer drivers. Further evaluation of a selected subset of these genes via quantitative reverse transcription-polymerase chain reaction determined that some genes exhibited similar regulation by pegvisomant in prostate cancer cells whether treatment was in vivo or in vitro, indicating direct regulation by GH via GHR activation in prostate cancer cells, whereas other genes responded to pegvisomant only in vivo, suggesting indirect regulation by pegvisomant effects on the host endocrine environment. Similar results were observed for a prostate cancer cell line derived from the mouse transgenic adenocarcinoma of the mouse prostate (TRAMP) model.

The landscape of antibody binding in SARS-CoV-2 infection

PLoS Biology · 2021 · 89 citations

• Biology
• Virology
• Immunology

The search for potential antibody-based diagnostics, vaccines, and therapeutics for pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has focused almost exclusively on the spike (S) and nucleocapsid (N) proteins. Coronavirus membrane (M), ORF3a, and ORF8 proteins are humoral immunogens in other coronaviruses (CoVs) but remain largely uninvestigated for SARS-CoV-2. Here, we use ultradense peptide microarray mapping to show that SARS-CoV-2 infection induces robust antibody responses to epitopes throughout the SARS-CoV-2 proteome, particularly in M, in which 1 epitope achieved excellent diagnostic accuracy. We map 79 B cell epitopes throughout the SARS-CoV-2 proteome and demonstrate that antibodies that develop in response to SARS-CoV-2 infection bind homologous peptide sequences in the 6 other known human CoVs. We also confirm reactivity against 4 of our top-ranking epitopes by enzyme-linked immunosorbent assay (ELISA). Illness severity correlated with increased reactivity to 9 SARS-CoV-2 epitopes in S, M, N, and ORF3a in our population. Our results demonstrate previously unknown, highly reactive B cell epitopes throughout the full proteome of SARS-CoV-2 and other CoV proteins.

Distinct hypertrophic cardiomyopathy genotypes result in convergent sarcomeric proteoform profiles revealed by top-down proteomics

Proceedings of the National Academy of Sciences · 2020 · 107 citations

• Biology
• Genetics
• Bioinformatics

= 16). We observed a complex landscape of sarcomeric proteoforms arising from combinatorial PTMs, alternative splicing, and genetic variation in HCM. A coordinated decrease of phosphorylation in important myofilament and Z-disk proteins with a linear correlation suggests PTM cross-talk in the sarcomere and dysregulation of protein kinase A pathways in HCM. Strikingly, we discovered that the sarcomeric proteoform alterations in the myocardium of HCM patients undergoing septal myectomy were remarkably consistent, regardless of the underlying HCM-causing mutations. This study suggests that the manifestation of severe HCM coalesces at the proteoform level despite distinct genotype, which underscores the importance of molecular characterization of HCM phenotype and presents an opportunity to identify broad-spectrum treatments to mitigate the most severe manifestations of this genetically heterogenous disease.