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Nature Nov 2023During nutrient stress, macroautophagy degrades cellular macromolecules, thereby providing biosynthetic building blocks while simultaneously remodelling the proteome....
During nutrient stress, macroautophagy degrades cellular macromolecules, thereby providing biosynthetic building blocks while simultaneously remodelling the proteome. Although the machinery responsible for initiation of macroautophagy has been well characterized, our understanding of the extent to which individual proteins, protein complexes and organelles are selected for autophagic degradation, and the underlying targeting mechanisms, is limited. Here we use orthogonal proteomic strategies to provide a spatial proteome census of autophagic cargo during nutrient stress in mammalian cells. We find that macroautophagy has selectivity for recycling membrane-bound organelles (principally Golgi and endoplasmic reticulum). Through autophagic cargo prioritization, we identify a complex of membrane-embedded proteins, YIPF3 and YIPF4, as receptors for Golgiphagy. During nutrient stress, YIPF3 and YIPF4 interact with ATG8 proteins through LIR motifs and are mobilized into autophagosomes that traffic to lysosomes in a process that requires the canonical autophagic machinery. Cells lacking YIPF3 or YIPF4 are selectively defective in elimination of a specific cohort of Golgi membrane proteins during nutrient stress. Moreover, YIPF3 and YIPF4 play an analogous role in Golgi remodelling during programmed conversion of stem cells to the neuronal lineage in vitro. Collectively, the findings of this study reveal prioritization of membrane protein cargo during nutrient-stress-dependent proteome remodelling and identify a Golgi remodelling pathway that requires membrane-embedded receptors.
Topics: Animals; Autophagy; Autophagy-Related Protein 8 Family; Endoplasmic Reticulum; Golgi Apparatus; Mammals; Membrane Proteins; Nutrients; Proteome; Proteomics
PubMed: 37757899
DOI: 10.1038/s41586-023-06657-6 -
Proteome profiling of early gestational plasma reveals novel biomarkers of congenital heart disease.EMBO Molecular Medicine Dec 2023Prenatal diagnosis of congenital heart disease (CHD) relies primarily on fetal echocardiography conducted at mid-gestational age-the sensitivity of which varies among...
Prenatal diagnosis of congenital heart disease (CHD) relies primarily on fetal echocardiography conducted at mid-gestational age-the sensitivity of which varies among centers and practitioners. An objective method for early diagnosis is needed. Here, we conducted a case-control study recruiting 103 pregnant women with healthy offspring and 104 cases with CHD offspring, including VSD (42/104), ASD (20/104), and other CHD phenotypes. Plasma was collected during the first trimester and proteomic analysis was performed. Principal component analysis revealed considerable differences between the controls and the CHDs. Among the significantly altered proteins, 25 upregulated proteins in CHDs were enriched in amino acid metabolism, extracellular matrix receptor, and actin skeleton regulation, whereas 49 downregulated proteins were enriched in carbohydrate metabolism, cardiac muscle contraction, and cardiomyopathy. The machine learning model reached an area under the curve of 0.964 and was highly accurate in recognizing CHDs. This study provides a highly valuable proteomics resource to better recognize the cause of CHD and has developed a reliable objective method for the early recognition of CHD, facilitating early intervention and better prognosis.
Topics: Pregnancy; Humans; Female; Proteome; Case-Control Studies; Proteomics; Heart Defects, Congenital; Biomarkers; Cisplatin; Cyclophosphamide
PubMed: 37840432
DOI: 10.15252/emmm.202317745 -
Cell Reports. Medicine Dec 2023Chemoradiation and targeted therapies are the major treatments for colorectal cancer (CRC); however, molecular properties associated with therapy resistance are...
Chemoradiation and targeted therapies are the major treatments for colorectal cancer (CRC); however, molecular properties associated with therapy resistance are incompletely characterized. Here, we profile the proteome of 254 tumor tissues from patients with CRC undergoing chemotherapy, chemoradiation, or chemotherapy combined with targeted therapy. Proteome-based classification reveals four subtypes featured with distinct biological and therapeutic characteristics. The integrative analysis of CRC cell lines and clinical samples indicates that immune regulation is significantly associated with drug sensitivity. HSF1 can increase DNA damage repair and cell cycle, thus inducing resistance to radiation, while high expression of HDAC6 is negatively associated with response of cetuximab. Furthermore, we develop prognostic models with high accuracy to predict the therapeutic response, further validated by parallel reaction monitoring (PRM) assay in an independent validation cohort. This study provides a rich resource for investigating the mechanisms and indicators of chemoradiation and targeted therapy in CRC.
Topics: Humans; Colorectal Neoplasms; Proteomics; Proteome; Cetuximab; Prognosis
PubMed: 38086380
DOI: 10.1016/j.xcrm.2023.101311 -
Gastroenterology Jul 2023Colonic adenomatous polyps, or adenomas, are frequent precancerous lesions and the origin of most cases of colorectal adenocarcinoma. However, we know from epidemiologic...
BACKGROUND & AIMS
Colonic adenomatous polyps, or adenomas, are frequent precancerous lesions and the origin of most cases of colorectal adenocarcinoma. However, we know from epidemiologic studies that although most colorectal cancers (CRCs) originate from adenomas, only a small fraction of adenomas (3%-5%) ever progress to cancer. At present, there are no molecular markers to guide follow-up surveillance programs.
METHODS
We profiled, by mass spectrometry-based proteomics combined with machine learning analysis, a selected cohort of formalin-fixed, paraffin-embedded high-grade (HG) adenomas with long clinical follow-up, collected as part of the Danish national screening program. We grouped subjects in the cohort according to their subsequent history of findings: a nonmetachronous advanced neoplasia group (G0), with no new HG adenomas or CRCs up to 10 years after polypectomy, and a metachronous advanced neoplasia group (G1) where individuals developed a new HG adenoma or CRC within 5 years of diagnosis.
RESULTS
We generated a proteome dataset from 98 selected HG adenoma samples, including 20 technical replicates, of which 45 samples belonged to the nonmetachronous advanced neoplasia group and 53 to the metachronous advanced neoplasia group. The clear distinction of these 2 groups seen in a uniform manifold approximation and projection plot indicated that the information contained within the abundance of the ∼5000 proteins was sufficient to predict the future occurrence of HG adenomas or development of CRC.
CONCLUSIONS
We performed an in-depth analysis of quantitative proteomic data from 98 resected adenoma samples using various novel algorithms and statistical packages and found that their proteome can predict development of metachronous advanced lesions and progression several years in advance.
Topics: Humans; Proteome; Proteomics; Colorectal Neoplasms; Colonic Polyps; Adenoma; Neoplasms, Second Primary; Colonoscopy; Risk Factors
PubMed: 36966943
DOI: 10.1053/j.gastro.2023.03.208 -
Expert Reviews in Molecular Medicine Apr 2024Target deconvolution can help understand how compounds exert therapeutic effects and can accelerate drug discovery by helping optimise safety and efficacy, revealing... (Review)
Review
Target deconvolution can help understand how compounds exert therapeutic effects and can accelerate drug discovery by helping optimise safety and efficacy, revealing mechanisms of action, anticipate off-target effects and identifying opportunities for therapeutic expansion. Chemoproteomics, a combination of chemical biology with mass spectrometry has transformed target deconvolution. This review discusses modification-free chemoproteomic approaches that leverage the change in protein thermodynamics induced by small molecule ligand binding. Unlike modification-based methods relying on enriching specific protein targets, these approaches offer proteome-wide evaluations, driven by advancements in mass spectrometry sensitivity, increasing proteome coverage and quantitation methods. Advances in methods based on denaturation/precipitation by thermal or chemical denaturation, or by protease degradation are evaluated, emphasising the evolving landscape of chemoproteomics and its potential impact on future drug-development strategies.
Topics: Humans; Proteome; Drug Discovery; Mass Spectrometry; Drug Development
PubMed: 38604802
DOI: 10.1017/erm.2024.6 -
Cell Systems Nov 2023Spatial proteomics combining microscopy-based cell phenotyping with ultrasensitive mass-spectrometry-based proteomics is an emerging and powerful concept to study cell...
Spatial proteomics combining microscopy-based cell phenotyping with ultrasensitive mass-spectrometry-based proteomics is an emerging and powerful concept to study cell function and heterogeneity in (patho)physiology. However, optimized workflows that preserve morphological information for phenotype discovery and maximize proteome coverage of few or even single cells from laser microdissected tissue are currently lacking. Here, we report a robust and scalable workflow for the proteomic analysis of ultra-low-input archival material. Benchmarking in murine liver resulted in up to 2,000 quantified proteins from single hepatocyte contours and nearly 5,000 proteins from 50-cell regions. Applied to human tonsil, we profiled 146 microregions including T and B lymphocyte niches and quantified cell-type-specific markers, cytokines, and transcription factors. These data also highlighted proteome dynamics within activated germinal centers, illuminating sites undergoing B cell proliferation and somatic hypermutation. This approach has broad implications in biomedicine, including early disease profiling and drug target and biomarker discovery. A record of this paper's transparent peer review process is included in the supplemental information.
Topics: Humans; Animals; Mice; Proteome; Proteomics; Mass Spectrometry
PubMed: 37909047
DOI: 10.1016/j.cels.2023.10.003 -
Molecular & Cellular Proteomics : MCP Sep 2023Ribosome profiling (Ribo-Seq) has proven transformative for our understanding of the human genome and proteome by illuminating thousands of noncanonical sites of...
Ribosome profiling (Ribo-Seq) has proven transformative for our understanding of the human genome and proteome by illuminating thousands of noncanonical sites of ribosome translation outside the currently annotated coding sequences (CDSs). A conservative estimate suggests that at least 7000 noncanonical ORFs are translated, which, at first glance, has the potential to expand the number of human protein CDSs by 30%, from ∼19,500 annotated CDSs to over 26,000 annotated CDSs. Yet, additional scrutiny of these ORFs has raised numerous questions about what fraction of them truly produce a protein product and what fraction of those can be understood as proteins according to conventional understanding of the term. Adding further complication is the fact that published estimates of noncanonical ORFs vary widely by around 30-fold, from several thousand to several hundred thousand. The summation of this research has left the genomics and proteomics communities both excited by the prospect of new coding regions in the human genome but searching for guidance on how to proceed. Here, we discuss the current state of noncanonical ORF research, databases, and interpretation, focusing on how to assess whether a given ORF can be said to be "protein coding."
Topics: Humans; Proteome; Protein Biosynthesis; Proteomics; Ribosome Profiling; Ribosomes; Open Reading Frames
PubMed: 37572790
DOI: 10.1016/j.mcpro.2023.100631 -
Journal of Proteome Research Oct 2023We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data-independent acquisition, the Thermo Scientific...
We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data-independent acquisition, the Thermo Scientific Orbitrap Astral mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific Orbitrap mass spectrometers, which have long been the gold standard for high-resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high-quality quantitative measurements across a wide dynamic range. We also use a newly developed extracellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5000 plasma proteins in a 60 min gradient with the Orbitrap Astral mass spectrometer.
Topics: Proteomics; Mass Spectrometry; Peptides; Proteome; Blood Proteins
PubMed: 37683181
DOI: 10.1021/acs.jproteome.3c00357 -
Cell Reports Nov 2023Plasmodium parasites contribute to one of the highest global infectious disease burdens. To achieve this success, the parasite has evolved a range of specialized...
Plasmodium parasites contribute to one of the highest global infectious disease burdens. To achieve this success, the parasite has evolved a range of specialized subcellular compartments to extensively remodel the host cell for its survival. The information to fully understand these compartments is likely hidden in the so far poorly characterized Plasmodium species spatial proteome. To address this question, we determined the steady-state subcellular location of more than 12,000 parasite proteins across five different species by extensive subcellular fractionation of erythrocytes infected by Plasmodium falciparum, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. This comparison of the pan-species spatial proteomes and their expression patterns indicates increasing species-specific proteins associated with the more external compartments, supporting host adaptations and post-transcriptional regulation. The spatial proteome offers comprehensive insight into the different human, simian, and rodent Plasmodium species, establishing a powerful resource for understanding species-specific host adaptation processes in the parasite.
Topics: Humans; Proteomics; Malaria; Proteome; Plasmodium berghei; Erythrocytes
PubMed: 37952150
DOI: 10.1016/j.celrep.2023.113419 -
Molecular & Cellular Proteomics : MCP Aug 2023Protein aggregation of amyloid-β peptides and tau are pathological hallmarks of Alzheimer's disease (AD), which are often resistant to detergent extraction and thus... (Meta-Analysis)
Meta-Analysis
Protein aggregation of amyloid-β peptides and tau are pathological hallmarks of Alzheimer's disease (AD), which are often resistant to detergent extraction and thus enriched in the insoluble proteome. However, additional proteins that coaccumulate in the detergent-insoluble AD brain proteome remain understudied. Here, we comprehensively characterized key proteins and pathways in the detergent-insoluble proteome from human AD brain samples using differential extraction, tandem mass tag (TMT) labeling, and two-dimensional LC-tandem mass spectrometry. To improve quantification accuracy of the TMT method, we developed a complement TMT-based strategy to correct for ratio compression. Through the meta-analysis of two independent detergent-insoluble AD proteome datasets (8914 and 8917 proteins), we identified 190 differentially expressed proteins in AD compared with control brains, highlighting the pathways of amyloid cascade, RNA splicing, endocytosis/exocytosis, protein degradation, and synaptic activity. To differentiate the truly detergent-insoluble proteins from copurified background during protein extraction, we analyzed the fold of enrichment for each protein by comparing the detergent-insoluble proteome with the whole proteome from the same AD samples. Among the 190 differentially expressed proteins, 84 (51%) proteins of the upregulated proteins (n = 165) were enriched in the insoluble proteome, whereas all downregulated proteins (n = 25) were not enriched, indicating that they were copurified components. The vast majority of these enriched 84 proteins harbor low-complexity regions in their sequences, including amyloid-β, Tau, TARDBP/TAR DNA-binding protein 43, SNRNP70/U1-70K, MDK, PTN, NTN1, NTN3, and SMOC1. Moreover, many of the enriched proteins in AD were validated in the detergent-insoluble proteome by five steps of differential extraction, proteomic analysis, or immunoblotting. Our study reveals a resource list of proteins and pathways that are exclusively present in the detergent-insoluble proteome, providing novel molecular insights to the formation of protein pathology in AD.
Topics: Humans; Alzheimer Disease; Proteome; Detergents; Proteomics; Tandem Mass Spectrometry; Brain; Ribonucleoprotein, U1 Small Nuclear
PubMed: 37356496
DOI: 10.1016/j.mcpro.2023.100608