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Nature Methods Oct 2023Single-cell proteomics by mass spectrometry is emerging as a powerful and unbiased method for the characterization of biological heterogeneity. So far, it has been...
Single-cell proteomics by mass spectrometry is emerging as a powerful and unbiased method for the characterization of biological heterogeneity. So far, it has been limited to cultured cells, whereas an expansion of the method to complex tissues would greatly enhance biological insights. Here we describe single-cell Deep Visual Proteomics (scDVP), a technology that integrates high-content imaging, laser microdissection and multiplexed mass spectrometry. scDVP resolves the context-dependent, spatial proteome of murine hepatocytes at a current depth of 1,700 proteins from a cell slice. Half of the proteome was differentially regulated in a spatial manner, with protein levels changing dramatically in proximity to the central vein. We applied machine learning to proteome classes and images, which subsequently inferred the spatial proteome from imaging data alone. scDVP is applicable to healthy and diseased tissues and complements other spatial proteomics and spatial omics technologies.
Topics: Animals; Mice; Proteome; Mass Spectrometry; Proteomics; Laser Capture Microdissection
PubMed: 37783884
DOI: 10.1038/s41592-023-02007-6 -
Nature Communications Jan 2023A plethora of software suites and multiple classes of spectral libraries have been developed to enhance the depth and robustness of data-independent acquisition (DIA)...
A plethora of software suites and multiple classes of spectral libraries have been developed to enhance the depth and robustness of data-independent acquisition (DIA) data processing. However, how the combination of a DIA software tool and a spectral library impacts the outcome of DIA proteomics and phosphoproteomics data analysis has been rarely investigated using benchmark data that mimics biological complexity. In this study, we create DIA benchmark data sets simulating the regulation of thousands of proteins in a complex background, which are collected on both an Orbitrap and a timsTOF instruments. We evaluate four commonly used software suites (DIA-NN, Spectronaut, MaxDIA and Skyline) combined with seven different spectral libraries in global proteome analysis. Moreover, we assess their performances in analyzing phosphopeptide standards and TNF-α-induced phosphoproteome regulation. Our study provides a practical guidance on how to construct a robust data analysis pipeline for different proteomics studies implementing the DIA technique.
Topics: Proteomics; Benchmarking; Workflow; Mass Spectrometry; Software; Proteome
PubMed: 36609502
DOI: 10.1038/s41467-022-35740-1 -
Molecular & Cellular Proteomics : MCP Jan 2020The main challenge of bottom-up proteomic sample preparation is to extract proteomes in a manner that enables efficient protein digestion for subsequent mass...
The main challenge of bottom-up proteomic sample preparation is to extract proteomes in a manner that enables efficient protein digestion for subsequent mass spectrometric analysis. Today's sample preparation strategies are commonly conceptualized around the removal of detergents, which are essential for extraction but strongly interfere with digestion and LC-MS. These multi-step preparations contribute to a lack of reproducibility as they are prone to losses, biases and contaminations, while being time-consuming and labor-intensive. We report a detergent-free method, named Sample Preparation by Easy Extraction and Digestion (SPEED), which consists of three mandatory steps, acidification, neutralization and digestion. SPEED is a universal method for peptide generation from various sources and is easily applicable even for lysis-resistant sample types as pure trifluoroacetic acid (TFA) is used for highly efficient protein extraction by complete sample dissolution. The protocol is highly reproducible, virtually loss-less, enables very rapid sample processing and is superior to the detergent/chaotropic agent-based methods FASP, ISD-Urea and SP3 for quantitative proteomics. SPEED holds the potential to dramatically simplify and standardize sample preparation while improving the depth of proteome coverage especially for challenging samples.
Topics: Animals; Bacillus cereus; Chromatography, Liquid; Detergents; Escherichia coli K12; HeLa Cells; Humans; Lung; Mice; Mice, Inbred C57BL; Peptides; Proteins; Proteolysis; Proteome; Proteomics; Reproducibility of Results; Staphylococcus aureus; Tandem Mass Spectrometry; Trifluoroacetic Acid; Urea
PubMed: 31754045
DOI: 10.1074/mcp.TIR119.001616 -
Annual Review of Genomics and Human... Aug 2022Proteins are the molecular effectors of the information encoded in the genome. Proteomics aims at understanding the molecular functions of proteins in their biological... (Review)
Review
Proteins are the molecular effectors of the information encoded in the genome. Proteomics aims at understanding the molecular functions of proteins in their biological context. In contrast to transcriptomics and genomics, the study of proteomes provides deeper insight into the dynamic regulatory layers encoded at the protein level, such as posttranslational modifications, subcellular localization, cell signaling, and protein-protein interactions. Currently, mass spectrometry (MS)-based proteomics is the technology of choice for studying proteomes at a system-wide scale, contributing to clinical biomarker discovery and fundamental molecular biology. MS technologies are continuously being developed to fulfill the requirements of speed, resolution, and quantitative accuracy, enabling the acquisition of comprehensive proteomes. In this review, we present how MS technology and acquisition methods have evolved to meet the requirements of cutting-edge proteomics research, which is describing the human proteome and its dynamic posttranslational modifications with unprecedented depth. Finally, we provide a perspective on studying proteomes at single-cell resolution.
Topics: Genome; Humans; Mass Spectrometry; Protein Processing, Post-Translational; Proteome; Proteomics
PubMed: 35440146
DOI: 10.1146/annurev-genom-112921-024948 -
Annual Review of Virology Sep 2021The abundance, localization, modifications, and protein-protein interactions of many host cell and virus proteins can change dynamically throughout the course of any... (Review)
Review
The abundance, localization, modifications, and protein-protein interactions of many host cell and virus proteins can change dynamically throughout the course of any viral infection. Studying these changes is critical for a comprehensive understanding of how viruses replicate and cause disease, as well as for the development of antiviral therapeutics and vaccines. Previously, we developed a mass spectrometry-based technique called quantitative temporal viromics (QTV), which employs isobaric tandem mass tags (TMTs) to allow precise comparative quantification of host and virus proteomes through a whole time course of infection. In this review, we discuss the utility and applications of QTV, exemplified by numerous studies that have since used proteomics with a variety of quantitative techniques to study virus infection through time.
Topics: Mass Spectrometry; Proteome; Proteomics; Viral Proteins; Viruses
PubMed: 34129369
DOI: 10.1146/annurev-virology-091919-104458 -
Aging Jun 2020Skin aging is a specific manifestation of the physiological aging process that occurs in virtually all organisms. In this study, we used data independent acquisition... (Comparative Study)
Comparative Study Observational Study
Skin aging is a specific manifestation of the physiological aging process that occurs in virtually all organisms. In this study, we used data independent acquisition mass spectrometry to perform a comparative analysis of protein expression in volar forearm skin samples from of 20 healthy young and elderly Chinese individuals. Our quantitative proteomic analysis identified a total of 95 differentially expressed proteins (DEPs) in aged skin compared to young skin. Enrichment analyses of these DEPs (57 upregulated and 38 downregulated proteins) based on the GO, KEGG, and KOG databases revealed functional clusters associated with immunity and inflammation, oxidative stress, biosynthesis and metabolism, proteases, cell proliferation, cell differentiation, and apoptosis. We also found that GAPDH, which was downregulated in aged skin samples, was the top hub gene in a protein-protein interaction network analysis. Some of the DEPs identified herein had been previously correlated with aging of the skin and other organs, while others may represent novel age-related entities. Our non-invasive proteomics analysis of human epidermal proteins may guide future research on skin aging to help develop treatments for age-related skin conditions and rejuvenation.
Topics: Adult; Age Factors; Aged; Asian People; Down-Regulation; Epidermis; Forearm; Healthy Volunteers; Humans; Mass Spectrometry; Middle Aged; Principal Component Analysis; Protein Interaction Mapping; Protein Interaction Maps; Proteome; Proteomics; Skin; Skin Aging; Up-Regulation; Young Adult
PubMed: 32602849
DOI: 10.18632/aging.103461 -
Scientific Reports Aug 2020Cancer cells release small extracellular vesicles, exosomes, that have been shown to contribute to various aspects of cancer development and progression. Differential...
Cancer cells release small extracellular vesicles, exosomes, that have been shown to contribute to various aspects of cancer development and progression. Differential analysis of exosomal proteomes from cancerous and non-tumorigenic breast cell lines can provide valuable information related to breast cancer progression and metastasis. Moreover, such a comparison can be explored to find potentially new protein biomarkers for early disease detection. In this study, exosomal proteomes of MDA-MB-231, a metastatic breast cancer cell line, and MCF-10A, a non-cancerous epithelial breast cell line, were identified by nano-liquid chromatography coupled to tandem mass spectrometry. We also tested three exosomes isolation methods (ExoQuick, Ultracentrifugation (UC), and Ultrafiltration-Ultracentrifugation) and detergents (n-dodecyl β-D-maltoside, Triton X-100, and Digitonin) for solubilization of exosomal proteins and enhanced detection by mass spectrometry. A total of 1,107 exosomal proteins were identified in both cell lines, 726 of which were unique to the MDA-MB-231 breast cancer cell line. Among them, 87 proteins were predicted to be relevant to breast cancer and 16 proteins to cancer metastasis. Three exosomal membrane/surface proteins, glucose transporter 1 (GLUT-1), glypican 1 (GPC-1), and disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), were identified as potential breast cancer biomarkers and validated with Western blotting and high-resolution flow cytometry. We demonstrated that exosomes are a rich source of breast cancer-related proteins and surface biomarkers that may be used for disease diagnosis and prognosis.
Topics: Biomarkers, Tumor; Breast Neoplasms; Exosomes; Female; Humans; Mass Spectrometry; Proteome; Proteomics; Tumor Cells, Cultured; Ultracentrifugation
PubMed: 32782317
DOI: 10.1038/s41598-020-70393-4 -
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 -
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 -
International Journal of Molecular... Nov 2022Inherited metabolic disorders (IMD) are rare medical conditions caused by genetic defects that interfere with the body's metabolism. The clinical phenotype is highly... (Review)
Review
Inherited metabolic disorders (IMD) are rare medical conditions caused by genetic defects that interfere with the body's metabolism. The clinical phenotype is highly variable and can present at any age, although it more often manifests in childhood. The number of treatable IMDs has increased in recent years, making early diagnosis and a better understanding of the natural history of the disease more important than ever. In this review, we discuss the main challenges faced in applying proteomics to the study of IMDs, and the key advances achieved in this field using tandem mass spectrometry (MS/MS). This technology enables the analysis of large numbers of proteins in different body fluids (serum, plasma, urine, saliva, tears) with a single analysis of each sample, and can even be applied to dried samples. MS/MS has thus emerged as the tool of choice for proteome characterization and has provided new insights into many diseases and biological systems. In the last 10 years, sequential window acquisition of all theoretical fragmentation spectra mass spectrometry (SWATH-MS) has emerged as an accurate, high-resolution technique for the identification and quantification of proteins differentially expressed between healthy controls and IMD patients. Proteomics is a particularly promising approach to help obtain more information on rare genetic diseases, including identification of biomarkers to aid early diagnosis and better understanding of the underlying pathophysiology to guide the development of new therapies. Here, we summarize new and emerging proteomic technologies and discuss current uses and limitations of this approach to identify and quantify proteins. Moreover, we describe the use of proteomics to identify the mechanisms regulating complex IMD phenotypes; an area of research essential to better understand these rare disorders and many other human diseases.
Topics: Humans; Proteomics; Tandem Mass Spectrometry; Proteome; Biomarkers; Metabolic Diseases
PubMed: 36499071
DOI: 10.3390/ijms232314744