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Journal of the American Society For... Aug 2023Multiplexing enables the monitoring of hundreds to thousands of proteins in quantitative proteomics analyses and increases sample throughput. In most...
Multiplexing enables the monitoring of hundreds to thousands of proteins in quantitative proteomics analyses and increases sample throughput. In most mass-spectrometry-based proteomics workflows, multiplexing is achieved by labeling biological samples with heavy isotopes via precursor isotopic labeling or isobaric tagging. Enhanced multiplexing strategies, such as combined precursor isotopic labeling and isobaric tagging (cPILOT), combine multiple technologies to afford an even higher sample throughput. Critical to enhanced multiplexing analyses is ensuring that analytical performance is optimal and that missingness of sample channels is minimized. Automation of sample preparation steps and use of quality control (QC) metrics can be incorporated into multiplexing analyses and reduce the likelihood of missing information, thus maximizing the amount of usable quantitative data. Here, we implemented QC metrics previously developed in our laboratory to evaluate a 36-plex cPILOT experiment that encompassed 144 mouse samples of various tissue types, time points, genotypes, and biological replicates. The evaluation focuses on the use of a sample pool generated from all samples in the experiment to monitor the daily instrument performance and to provide a means for data normalization across sample batches. Our results show that tracking QC metrics enabled the quantification of ∼7000 proteins in each sample batch, of which ∼70% had minimal missing values across up to 36 sample channels. Implementation of QC metrics for future cPILOT studies as well as other enhanced multiplexing strategies will help yield high-quality data sets.
Topics: Mice; Animals; Proteins; Mass Spectrometry; Proteomics; Isotope Labeling; Quality Control; Proteome
PubMed: 37459602
DOI: 10.1021/jasms.3c00179 -
Proteomics Jun 2024Extracellular vesicles (EVs), the key players in inter-cellular communication, are produced by all cell types and are present in all body fluids. Analysis of the... (Review)
Review
Extracellular vesicles (EVs), the key players in inter-cellular communication, are produced by all cell types and are present in all body fluids. Analysis of the proteome content is an important approach in structural and functional studies of these vesicles. EVs circulating in human plasma are heterogeneous in size, cellular origin, and functions. This heterogeneity and the potential presence of contamination with plasma components such as lipoprotein particles and soluble plasma proteins represent a challenge in profiling the proteome of EV subsets by mass spectrometry. An immunocapture strategy prior to mass spectrometry may be used to isolate a homogeneous subpopulation of small EVs (sEV) with a specific endocytic origin from plasma or other biofluids. Immunocapture selectively separates EV subpopulations in biofluids based on the presence of a unique protein carried on the vesicle surface. The advantages and disadvantages of EV immune capture as a preparative step for mass spectrometry are discussed.
Topics: Humans; Extracellular Vesicles; Proteomics; Mass Spectrometry; Proteome; Blood Proteins
PubMed: 37713108
DOI: 10.1002/pmic.202300180 -
Nucleic Acids Research Jan 2024Advancements in mass spectrometry (MS)-based proteomics have greatly facilitated the large-scale quantification of proteins and microproteins, thereby revealing altered...
Advancements in mass spectrometry (MS)-based proteomics have greatly facilitated the large-scale quantification of proteins and microproteins, thereby revealing altered signalling pathways across many different cancer types. However, specialized and comprehensive resources are lacking for cancer proteomics. Here, we describe CancerProteome (http://bio-bigdata.hrbmu.edu.cn/CancerProteome), which functionally deciphers and visualizes the proteome landscape in cancer. We manually curated and re-analyzed publicly available MS-based quantification and post-translational modification (PTM) proteomes, including 7406 samples from 21 different cancer types, and also examined protein abundances and PTM levels in 31 120 proteins and 4111 microproteins. Six major analytical modules were developed with a view to describe protein contributions to carcinogenesis using proteome analysis, including conventional analyses of quantitative and the PTM proteome, functional enrichment, protein-protein associations by integrating known interactions with co-expression signatures, drug sensitivity and clinical relevance analyses. Moreover, protein abundances, which correlated with corresponding transcript or PTM levels, were evaluated. CancerProteome is convenient as it allows users to access specific proteins/microproteins of interest using quick searches or query options to generate multiple visualization results. In summary, CancerProteome is an important resource, which functionally deciphers the cancer proteome landscape and provides a novel insight for the identification of tumor protein markers in cancer.
Topics: Humans; Mass Spectrometry; Neoplasms; Protein Processing, Post-Translational; Proteome; Proteomics; Databases, Protein
PubMed: 37823596
DOI: 10.1093/nar/gkad824 -
The EMBO Journal Dec 2023Substantial efforts are underway to deepen our understanding of human brain morphology, structure, and function using high-resolution imaging as well as high-content...
Substantial efforts are underway to deepen our understanding of human brain morphology, structure, and function using high-resolution imaging as well as high-content molecular profiling technologies. The current work adds to these approaches by providing a comprehensive and quantitative protein expression map of 13 anatomically distinct brain regions covering more than 11,000 proteins. This was enabled by the optimization, characterization, and implementation of a high-sensitivity and high-throughput microflow liquid chromatography timsTOF tandem mass spectrometry system (LC-MS/MS) capable of analyzing more than 2,000 consecutive samples prepared from formalin-fixed paraffin embedded (FFPE) material. Analysis of this proteomic resource highlighted brain region-enriched protein expression patterns and functional protein classes, protein localization differences between brain regions and individual markers for specific areas. To facilitate access to and ease further mining of the data by the scientific community, all data can be explored online in a purpose-built R Shiny app (https://brain-region-atlas.proteomics.ls.tum.de).
Topics: Humans; Chromatography, Liquid; Proteomics; Paraffin Embedding; Tandem Mass Spectrometry; Proteins; Brain; Proteome
PubMed: 37916885
DOI: 10.15252/embj.2023114665 -
Forensic Science International. Genetics Sep 2023Human hair is often found at crime scenes, persists for a long time, and is a valuable biological specimen in forensic investigations. Hair contains minimal intact...
Human hair is often found at crime scenes, persists for a long time, and is a valuable biological specimen in forensic investigations. Hair contains minimal intact nuclear DNA for the discrimination of individual identity. In such cases, proteomics evaluation of hair proteins could provide an attractive alternative for protein-based human identification. Therefore, this study adopted a proteomic approach to profile hair shafts from both males and females across different ethnic populations including Chinese, Indians, Malays, and Filipinos in their 20-80 s. First, hair proteins were extracted by different methods to adopt the most suitable protocol that produced the highest extraction efficiency based on most significant enrichment of keratins and keratin-associated proteins. Abundance of hair keratins including both types I and II, and keratin-associated proteins, estimated using label-free quantification, showed distinguishable profiles, and the possibilities of distinguishing individuals within each ethnic origin. Similarly, several protein candidates and their abundances could be used to distinguish sex and age of individuals. This study explored the possibility of utilizing hair proteomics phenotyping in forensic science to differentiate individuals across various ethnic groups, sex and age.
Topics: Male; Female; Humans; Proteome; Proteomics; Keratins; Hair; Demography
PubMed: 37482024
DOI: 10.1016/j.fsigen.2023.102914 -
Life Science Alliance Sep 2023Mitochondrial dysfunction and cellular senescence are hallmarks of aging. However, the relationship between these two phenomena remains incompletely understood. In this...
Mitochondrial dysfunction and cellular senescence are hallmarks of aging. However, the relationship between these two phenomena remains incompletely understood. In this study, we investigated the rewiring of mitochondria upon development of the senescent state in human IMR90 fibroblasts. Determining the bioenergetic activities and abundance of mitochondria, we demonstrate that senescent cells accumulate mitochondria with reduced OXPHOS activity, resulting in an overall increase of mitochondrial activities in senescent cells. Time-resolved proteomic analyses revealed extensive reprogramming of the mitochondrial proteome upon senescence development and allowed the identification of metabolic pathways that are rewired with different kinetics upon establishment of the senescent state. Among the early responding pathways, the degradation of branched-chain amino acid was increased, whereas the one carbon folate metabolism was decreased. Late-responding pathways include lipid metabolism and mitochondrial translation. These signatures were confirmed by metabolic flux analyses, highlighting metabolic rewiring as a central feature of mitochondria in cellular senescence. Together, our data provide a comprehensive view on the changes in mitochondrial proteome in senescent cells and reveal how the mitochondrial metabolism is rewired in senescent cells.
Topics: Humans; Proteomics; Proteome; Mitochondria; Aging; Cellular Senescence
PubMed: 37321846
DOI: 10.26508/lsa.202302127 -
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 -
Nature Communications Sep 2023Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain...
Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain obscured with population-based techniques. The limited amount of material encapsulated in a single cell however, raises significant technical challenges to molecular profiling. Due to extensive optimization efforts, single-cell proteomics by Mass Spectrometry (scp-MS) has emerged as a powerful tool to facilitate proteome profiling from ultra-low amounts of input, although further development is needed to realize its full potential. To this end, we carry out comprehensive analysis of orbitrap-based data-independent acquisition (DIA) for limited material proteomics. Notably, we find a fundamental difference between optimal DIA methods for high- and low-load samples. We further improve our low-input DIA method by relying on high-resolution MS1 quantification, thus enhancing sensitivity by more efficiently utilizing available mass analyzer time. With our ultra-low input tailored DIA method, we are able to accommodate long injection times and high resolution, while keeping the scan cycle time low enough to ensure robust quantification. Finally, we demonstrate the capability of our approach by profiling mouse embryonic stem cell culture conditions, showcasing heterogeneity in global proteomes and highlighting distinct differences in key metabolic enzyme expression in distinct cell subclusters.
Topics: Animals; Mice; Proteomics; Mass Spectrometry; Mouse Embryonic Stem Cells; Proteome; Single-Cell Analysis
PubMed: 37737208
DOI: 10.1038/s41467-023-41602-1 -
Frontiers in Endocrinology 2023There are no validated clinical or laboratory biomarkers to identify and differentiate endotypes of type 1 diabetes (T1D) or the risk of progression to chronic...
INTRODUCTION
There are no validated clinical or laboratory biomarkers to identify and differentiate endotypes of type 1 diabetes (T1D) or the risk of progression to chronic complications. Extracellular vesicles (EVs) have been studied as biomarkers in several different disease states but have not been well studied in T1D.
METHODS
As the initial step towards circulating biomarker identification in T1D, this pilot study aimed to provide an initial characterization of the proteomic and phosphoproteomic landscape of circulating EV-enriched preparations in participants with established T1D (N=10) and healthy normal volunteers (Controls) (N=7) (NCT03379792) carefully matched by age, race/ethnicity, sex, and BMI. EV-enriched preparations were obtained using EVtrap technology. Proteins were identified and quantified by LC-MS analysis. Differential abundance and coexpression network (WGCNA), and pathway enrichment analyses were implemented.
RESULTS
The detected proteins and phosphoproteins were enriched (75%) in exosomal proteins cataloged in the ExoCarta database. A total of 181 proteins and 8 phosphoproteins were differentially abundant in participants with T1D compared to controls, including some well-known EVproteins (i.e., CD63, RAB14, BSG, LAMP2, and EZR). Enrichment analyses of differentially abundant proteins and phosphoproteins of EV-enriched preparations identified associations with neutrophil, platelet, and immune response functions, as well as prion protein aggregation. Downregulated proteins were involved in MHC class II signaling and the regulation of monocyte differentiation. Potential key roles in T1D for C1q, plasminogen, IL6ST, CD40, HLA-DQB1, HLA-DRB1, CD74, NUCB1, and SAP, are highlighted. Remarkably, WGCNA uncovered two protein modules significantly associated with pancreas size, which may be implicated in the pathogenesis of T1D. Similarly, these modules showed significant enrichment for membrane compartments, processes associated with inflammation and the immune response, and regulation of viral processes, among others.
DISCUSSION
This study demonstrates the potential of proteomic and phosphoproteomic signatures of EV-enriched preparations to provide insight into the pathobiology of T1D. The WGCNA analysis could be a powerful tool to discriminate signatures associated with different pathobiological components of the disease.
Topics: Humans; Diabetes Mellitus, Type 1; Proteome; Proteomics; Pilot Projects; Biomarkers; Phosphoproteins; Extracellular Vesicles
PubMed: 37576973
DOI: 10.3389/fendo.2023.1219293 -
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