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Proteomics Jan 2024Increased throughput in proteomic experiments can improve accessibility of proteomic platforms, reduce costs, and facilitate new approaches in systems biology and...
Increased throughput in proteomic experiments can improve accessibility of proteomic platforms, reduce costs, and facilitate new approaches in systems biology and biomedical research. Here we propose combination of analytical flow rate chromatography with ion mobility separation of peptide ions, data-independent acquisition, and data analysis with the DIA-NN software suite, to achieve high-quality proteomic experiments from limited sample amounts, at a throughput of up to 400 samples per day. For instance, when benchmarking our workflow using a 500-μL/min flow rate and 3-min chromatographic gradients, we report the quantification of 5211 proteins from 2 μg of a mammalian cell-line standard at high quantitative accuracy and precision. We further used this platform to analyze blood plasma samples from a cohort of COVID-19 inpatients, using a 3-min chromatographic gradient and alternating column regeneration on a dual pump system. The method delivered a comprehensive view of the COVID-19 plasma proteome, allowing classification of the patients according to disease severity and revealing plasma biomarker candidates.
Topics: Animals; Humans; Proteomics; Peptides; Proteome; Chromatography, Liquid; COVID-19; Mammals
PubMed: 37287406
DOI: 10.1002/pmic.202300100 -
Scientific Reports Sep 2023Congenital diaphragmatic hernia (CDH) is a severe birth defect frequently associated with pulmonary hypoplasia, pulmonary hypertension, and heart failure. Since amniotic...
Congenital diaphragmatic hernia (CDH) is a severe birth defect frequently associated with pulmonary hypoplasia, pulmonary hypertension, and heart failure. Since amniotic fluid comprises proteins of both fetal and maternal origin, its analysis could provide insights on mechanisms underlying CDH and provide biomarkers for early diagnosis, severity of pulmonary changes and treatment response. The study objective was to identify proteomic changes in amniotic fluid consistently associated with CDH. Amniotic fluid was obtained at term (37-39 weeks) from women with normal pregnancies (n = 5) or carrying fetuses with CDH (n = 5). After immuno-depletion of the highest abundance proteins, off-line fractionation and high-resolution tandem mass spectrometry were performed and quantitative differences between the proteomes of the groups were determined. Of 1036 proteins identified, 218 were differentially abundant. Bioinformatics analysis showed significant changes in GP6 signaling, in the MSP-RON signaling in macrophages pathway and in networks associated with cardiovascular system development and function, connective tissue disorders and dermatological conditions. Differences in selected proteins, namely pulmonary surfactant protein B, osteopontin, kallikrein 5 and galectin-3 were validated by orthogonal testing using ELISA in larger cohorts and showed statistically significant differences aiding in the diagnosis and prediction of CDH. The findings provide potential tools for clinical management of CDH.
Topics: Pregnancy; Humans; Female; Hernias, Diaphragmatic, Congenital; Amniotic Fluid; Proteomics; Proteome; Biomarkers
PubMed: 37726509
DOI: 10.1038/s41598-023-42576-2 -
Nature Communications Dec 2023The analysis of proteins that are newly synthesized upon a cellular perturbation can provide detailed insight into the proteomic response that is elicited by specific...
The analysis of proteins that are newly synthesized upon a cellular perturbation can provide detailed insight into the proteomic response that is elicited by specific cues. This can be investigated by pulse-labeling of cells with clickable and stable-isotope-coded amino acids for the enrichment and mass spectrometric characterization of newly synthesized proteins (NSPs), however convoluted protocols prohibit their routine application. Here we report the optimization of multiple steps in sample preparation, mass spectrometry and data analysis, and we integrate them into a semi-automated workflow for the quantitative analysis of the newly synthesized proteome (QuaNPA). Reduced input requirements and data-independent acquisition (DIA) enable the analysis of triple-SILAC-labeled NSP samples, with enhanced throughput while featuring high quantitative accuracy. We apply QuaNPA to investigate the time-resolved cellular response to interferon-gamma (IFNg), observing rapid induction of targets 2 h after IFNg treatment. QuaNPA provides a powerful approach for large-scale investigation of NSPs to gain insight into complex cellular processes.
Topics: Proteome; Proteomics; Workflow; Amino Acids; Cell Line; Isotope Labeling
PubMed: 38086798
DOI: 10.1038/s41467-023-43919-3 -
Annals of Clinical and Translational... Nov 2023Amyotrophic lateral sclerosis (ALS) is a heterogeneous disease with a complex etiology that lacks biomarkers predicting disease progression. The objective of this study...
OBJECTIVE
Amyotrophic lateral sclerosis (ALS) is a heterogeneous disease with a complex etiology that lacks biomarkers predicting disease progression. The objective of this study was to use longitudinal cerebrospinal fluid (CSF) samples to identify biomarkers that distinguish fast progression (FP) from slow progression (SP) and assess their temporal response.
METHODS
We utilized mass spectrometry (MS)-based proteomics to identify candidate biomarkers using longitudinal CSF from a discovery cohort of SP and FP ALS patients. Immunoassays were used to quantify and validate levels of the top biomarkers. A state-transition mathematical model was created using the longitudinal MS data that also predicted FP versus SP.
RESULTS
We identified a total of 1148 proteins in the CSF of all ALS patients. Pathway analysis determined enrichment of pathways related to complement and coagulation cascades in FPs and synaptogenesis and glucose metabolism in SPs. Longitudinal analysis revealed a panel of 59 candidate markers that could segregate FP and SP ALS. Based on multivariate analysis, we identified three biomarkers (F12, RBP4, and SERPINA4) as top candidates that segregate ALS based on rate of disease progression. These proteins were validated in the discovery and a separate validation cohort. Our state-transition model determined that the overall variance of the proteome over time was predictive of the disease progression rate.
INTERPRETATION
We identified pathways and protein biomarkers that distinguish rate of ALS disease progression. A mathematical model of the CSF proteome determined that the change in entropy of the proteome over time was predictive of FP versus SP.
Topics: Humans; Amyotrophic Lateral Sclerosis; Proteome; Proteomics; Biomarkers; Disease Progression; Retinol-Binding Proteins, Plasma
PubMed: 37646115
DOI: 10.1002/acn3.51890 -
Angewandte Chemie (International Ed. in... Oct 2023Cellular proteins are dynamically regulated in response to environmental stimuli. Conventional proteomics compares the entire proteome in different cellular states to... (Review)
Review
Cellular proteins are dynamically regulated in response to environmental stimuli. Conventional proteomics compares the entire proteome in different cellular states to identify differentially expressed proteins, which suffers from limited sensitivity for analyzing acute and subtle changes. To address this challenge, nascent proteomics has been developed, which selectively analyzes the newly synthesized proteins, thus offering a more sensitive and timely insight into the dynamic changes of the proteome. In this Minireview, we discuss recent advancements in nascent proteomics, with an emphasis on methodological developments. Also, we delve into the current challenges and provide an outlook on the future prospects of this exciting field.
Topics: Proteome; Proteomics
PubMed: 37309018
DOI: 10.1002/anie.202305866 -
Developmental Cell Nov 2023Centrosomes are the major microtubule-organizing centers in animals and play fundamental roles in many cellular processes. Understanding how their composition varies...
Centrosomes are the major microtubule-organizing centers in animals and play fundamental roles in many cellular processes. Understanding how their composition varies across diverse cell types and how it is altered in disease are major unresolved questions, yet currently available centrosome isolation protocols are cumbersome and time-consuming, and they lack scalability. Here, we report the development of centrosome affinity capture (CAPture)-mass spectrometry (MS), a powerful one-step purification method to obtain high-resolution centrosome proteomes from mammalian cells. Utilizing a synthetic peptide derived from CCDC61 protein, CAPture specifically isolates intact centrosomes. Importantly, as a bead-based affinity method, it enables rapid sample processing and multiplexing unlike conventional approaches. Our study demonstrates the power of CAPture-MS to elucidate cell-type-dependent heterogeneity in centrosome composition, dissect hierarchical interactions, and identify previously unknown centrosome components. Overall, CAPture-MS represents a transformative tool to unveil temporal, regulatory, cell-type- and tissue-specific changes in centrosome proteomes in health and disease.
Topics: Animals; Proteomics; Proteome; Centrosome; Microtubule-Organizing Center; Microtubules; Mammals
PubMed: 37852252
DOI: 10.1016/j.devcel.2023.09.008 -
Pathologie (Heidelberg, Germany) Dec 2023Proteomics, the study of proteins and their functions, has greatly evolved due to advances in analytical chemistry and computational biology. Unlike genomics or... (Review)
Review
Proteomics, the study of proteins and their functions, has greatly evolved due to advances in analytical chemistry and computational biology. Unlike genomics or transcriptomics, proteomics captures the dynamic and diverse nature of proteins, which play crucial roles in cellular processes. This is exemplified in cancer, where genomic and transcriptomic information often falls short in reflecting actual protein expression and interactions. Liquid chromatography-mass spectrometry (LC-MS) is pivotal in proteomic data generation, enabling high-throughput analysis of protein samples. The MS-based workflow involves protein digestion, chromatographic separation, ionization, and fragmentation, leading to peptide identification and quantification. Computational biostatistics, particularly using tools in R (R Foundation for Statistical Computing, Vienna, Austria; www.R-project.org ), aid in data analysis, revealing protein expression patterns and correlations with clinical variables. Proteomic studies can be explorative, aiming to characterize entire proteomes, or targeted, focusing on specific proteins of interest. The integration of proteomics with genomics addresses database limitations and enhances peptide identification. Case studies in intrahepatic cholangiocarcinoma, glioblastoma multiforme, and pancreatic ductal adenocarcinoma highlight proteomics' clinical applications, from subtyping cancers to identifying diagnostic markers. Moreover, proteomic data augment molecular tumor boards by providing deeper insights into pathway activities and genomic mutations, supporting personalized treatment decisions. Overall, proteomics contributes significantly to advancing our understanding of cellular biology and improving clinical care.
Topics: Humans; Proteomics; Proteome; Peptides; Neoplasms; Computational Biology
PubMed: 37999758
DOI: 10.1007/s00292-023-01261-x -
Cancer Research Nov 2023Proteomics is a powerful approach that can rapidly enhance our understanding of cancer development. Detailed characterization of the genetic, pharmacogenomic, and immune...
UNLABELLED
Proteomics is a powerful approach that can rapidly enhance our understanding of cancer development. Detailed characterization of the genetic, pharmacogenomic, and immune landscape in relation to protein expression in patients with cancer could provide new insights into the functional roles of proteins in cancer. By taking advantage of the genotype data from The Cancer Genome Atlas and protein expression data from The Cancer Proteome Atlas, we characterized the effects of genetic variants on protein expression across 31 cancer types and identified approximately 100,000 protein quantitative trait loci (pQTL). Among these, over 8000 pQTLs were associated with patient overall survival. Furthermore, characterization of the impact of protein expression on more than 350 imputed anticancer drug responses in patients revealed nearly 230,000 significant associations. In addition, approximately 21,000 significant associations were identified between protein expression and immune cell abundance. Finally, a user-friendly data portal, GPIP (https://hanlaboratory.com/GPIP), was developed featuring multiple modules that enable researchers to explore, visualize, and browse multidimensional data. This detailed analysis reveals the associations between the proteomic landscape and genetic variation, patient outcome, the immune microenvironment, and drug response across cancer types, providing a resource that may offer valuable clinical insights and encourage further functional investigations of proteins in cancer.
SIGNIFICANCE
Comprehensive characterization of the relationship between protein expression and the genetic, pharmacogenomic, and immune landscape of tumors across cancer types provides a foundation for investigating the role of protein expression in cancer development and treatment.
Topics: Humans; Proteomics; Pharmacogenetics; Proteome; Genotype; Neoplasms; Tumor Microenvironment
PubMed: 37548539
DOI: 10.1158/0008-5472.CAN-23-0758 -
The Journal of Investigative Dermatology Aug 2023Hand eczema (HE) is a prevalent skin disease. However, the classification of HE into different subtypes remains challenging. A limited number of previous studies have...
Hand eczema (HE) is a prevalent skin disease. However, the classification of HE into different subtypes remains challenging. A limited number of previous studies have employed invasive biopsy-based strategies; yet, studies of the HE proteome using noninvasive tape-stripping methodology have not been reported. In this study, we wanted to assess whether global proteomic analysis of skin tape strip samples can be used for subclassification of patients with HE. Tape strips were collected from patients with HE and healthy skin. Liquid chromatography-mass spectrometry proteomics was performed, and the global protein expression was analyzed. We identified 2,919 proteins in stratum corneum-derived skin cells from tape strip samples. Compared with healthy skin, the lesional samples from patients with HE exhibited increased expression of immune-related markers and a decreased expression of structural barrier proteins. The difference between HE subtypes was restricted to the lesional skin areas and included an increased expression of skin barrier-related proteins independently of the concurrent AD. In conclusion, we found that the noninvasive tape strip method used in combination with liquid chromatography-mass spectrometry proteomics can be used for analysis of skin protein expression in patients with HE. Thus, the method shows potential for assessing the proteomic differences between subtypes of HE and biomarker discovery.
Topics: Humans; Proteome; Proteomics; Skin; Epidermis; Eczema; Biomarkers
PubMed: 36773646
DOI: 10.1016/j.jid.2022.12.024 -
Neuroscience Letters Sep 2023Spinal cord injury (SCI) is a devastating trauma of the central nervous system, with high levels of morbidity, disability, and mortality. To explore the underlying...
Spinal cord injury (SCI) is a devastating trauma of the central nervous system, with high levels of morbidity, disability, and mortality. To explore the underlying mechanism of SCI, we analyzed the proteome and phosphoproteome of rats at one week after SCI. We identified 465 up-regulated and 129 down-regulated differentially expressed proteins (DEPs), as well as 184 up-regulated and 40 down-regulated differentially expressed phosphoproteins (DEPPs). Using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis, we identified the biological characteristics of these proteins from the perspectives of cell component, biological process, and molecular function. We also found a lot of enriched functional pathways such as GABAergic synapse pathway, ErbB signaling pathway, tight junction, adherens junction. The integrated analysis of proteomics and phosphoproteomics yielded 22 differently expressed co-identified proteins of DEPs and DEPPs, which revealed strongly correlative patterns. These findings may help clarify the potential mechanisms of trauma and repair in SCI and may guide the development of novel treatments.
Topics: Rats; Animals; Gene Expression Profiling; Proteomics; Spinal Cord Injuries; Proteome; Spinal Cord
PubMed: 37597742
DOI: 10.1016/j.neulet.2023.137449