Comprehensive Protein Profiling Research Articles

Host-derived protein profiles of human neonatal meconium across gestational ages

Meconium, a non-invasive biomaterial reflecting prenatal substance accumulation, could provide valuable insights into neonatal health. However, the comprehensive protein profile of meconium across gestational ages remains unclear. Here, we conducted an extensive proteomic analysis of first meconium from 259 newborns across varied gestational ages to delineate protein composition and elucidate its relevance to neonatal diseases. The first meconium samples were collected, with the majority obtained before feeding, and the mean time for the first meconium passage from the anus was 11.9 ± 9.47 h. Our analysis revealed 5370 host-derived meconium proteins, which varied depending on sex and gestational age. Specifically, meconium from preterm infants exhibited elevated concentrations of proteins associated with the extracellular matrix. Additionally, the protein profiles of meconium also exhibited unique variations depending on both specific diseases, including gastrointestinal diseases, congenital heart diseases, and maternal conditions. Furthermore, we developed a machine learning model to predict gestational ages using meconium proteins. Our model suggests that newborns with gastrointestinal diseases and congenital heart diseases may have immature gastrointestinal systems. These findings highlight the intricate relationship between clinical parameters and meconium protein composition, offering potential for a novel approach to assess neonatal gastrointestinal health.

Abstract 75: Luciferase reporter cell lines allow simultaneous incorporation of tumor cells, innate immune cells, and adaptive immune cells for in-depth immune checkpoint studies

Abstract Despite the considerable success of immune checkpoint therapies targeting T cells, a sizable proportion of patients experience resistance or relapse due to the immunosuppressive nature of the tumor microenvironment. Myeloid cells, a major component that suppresses effector lymphocytes, have emerged as an alternative and promising therapeutic target. However, there is a deep lack of widely accessible immunological models capable of representing the intricate three-way interaction between tumor cells, T cells, and myeloid cells. To address this need, we conducted a comprehensive protein profiling of human tumor and immune cell lines available at ATCC for various established and novel immune checkpoint molecules. Cell lines with high endogenous expression of the immune checkpoint proteins, such as programmed death-ligand 1 and 2 (PD-L1 and PD-L2), cluster of differentiation 155 (CD155), B7 homolog 3 (B7-H3), sialic acid-binding Ig-like lectin 10 (Siglec-10), or signal-regulatory protein alpha (SIRPα), were selected and constructed into luciferase reporter cell lines. For tumor reporter cell lines, a gamma interferon activation site (GAS) response element was placed upstream of the luciferase gene in the lentiviral vector, enabling the activation of the JAK-STAT signaling pathway within tumor cells to induce luciferase expression. In myeloid reporter cell lines, a nuclear factor kappa B (NF-κB) response element replaced GAS to monitor the activation of the NF-κB signaling pathway. In the presence of corresponding immune checkpoint inhibitors that enhance T cell-mediated anti-tumor activity, these reporter cell lines produce a bioluminescent signal based on luciferase expression. This signal can be easily detected and quantified to assess the efficacy of the inhibitor. Our data revealed that bioluminescence intensity in the tumor and myeloid reporter cell lines increased by >100-fold in a dose-dependent manner in response to interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) stimulation, respectively, and by >50-fold in response to the conditioned media collected from activated primary T cells. Furthermore, in co-culture assays involving various combinations of immune cell and tumor cell types with corresponding immune checkpoint inhibitors, these reporter cell lines demonstrated a significant increase in bioluminescence intensity. In conclusion, these newly established luciferase reporter cell lines offer an excellent ex vivo model for cancer immunotherapy. These cell lines naturally express immune checkpoint proteins, enabling the sensitive and reproducible monitoring of combinatorial responses from various immune cell types. Citation Format: Hyeyoun Chang, Alicia C. Walker, John G. Foulke, Luping Chen, Fang Tian, Zhizhan Gu. Luciferase reporter cell lines allow simultaneous incorporation of tumor cells, innate immune cells, and adaptive immune cells for in-depth immune checkpoint studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 75.

Proteomic Profiling of Muscular Adaptations to Short-Term Concentric Versus Eccentric Exercise Training in Humans

The molecular mechanisms underlying muscular adaptations to concentric (CON) and eccentric (ECC) exercise training have been extensively explored. However, most previous studies have focused on specifically selected proteins, thus, unable to provide a comprehensive protein profile and potentially missing the crucial mechanisms underlying muscular adaptation to exercise training. We herein aimed to investigate proteomic profiles of human skeletal muscle in response to short-term resistance training. Twenty young males were randomly and evenly assigned to two groups to complete a 4-week either ECC or CON training program. Measurements of body composition and physiological function of the quadriceps femoris were conducted both before and after the training. Muscle biopsies from the vastus lateralis of randomly selected participants (five in ECC and four in CON) of both before and after the training were analyzed using the liquid-chromatography tandem mass spectrometry in combination with bioinformatics analysis. Neither group presented a significant difference in body composition or leg muscle mass; however, muscle peak torque, total work, and maximal voluntary contraction were significantly increased after the training in both groups. Proteomics analysis revealed 122 differentially abundant proteins (DAPs; p value < 0.05 & fold change >1.5 or <0.67) in ECC, of which the increased DAPs were mainly related to skeletal muscle contraction and cytoskeleton and enriched specifically in the pentose phosphate pathway, extracellular matrix–receptor interaction, and PI3K−Akt signaling pathway, whereas the decreased DAPs were associated with the mitochondrial respiratory chain. One hundred one DAPs were identified in CON, of which the increased DAPs were primarily involved in translation/protein synthesis and the mitochondria respiratory, whereas the decreased DAPs were related to metabolic processes, cytoskeleton, and de-ubiquitination. In conclusion, the 4-week CON and ECC training resulted in distinctly different proteomic profiles, especially in proteins related to muscular structure and metabolism.

Exploring the Molecular Tapestry: Organ-Specific Peptide and Protein Ultrafiltrates and Their Role in Therapeutics.

This study aims to characterize the proteome composition of organ-derived protein extracts from rabbits. Protein isolation was performed using soft homogenization and size exclusion via ultrafiltration. The proteome analysis of the ultrafiltrates was conducted using gel electrophoresis, and the mass spectrometry data were subjected to gene ontology analysis. Proteomic profiling revealed comprehensive protein profiles associated with RNA regulation, fatty acid binding, inflammatory response, oxidative stress, and metabolism. Additionally, our results demonstrate the presence of abundant small proteins, as observed in the mass spectrometry datasets. Small proteins and peptides are crucial in transcription modulation and various biological processes. The protein networks identified in the ultrafiltrates have the potential to enhance and complement biological therapeutic interventions. Data are available via ProteomeXchange with identifier PXD050039.

Breast cancer patient-derived microtumors resemble tumor heterogeneity and enable protein-based stratification and functional validation of individualized drug treatment

Despite tremendous progress in deciphering breast cancer at the genomic level, the pronounced intra- and intertumoral heterogeneity remains a major obstacle to the advancement of novel and more effective treatment approaches. Frequent treatment failure and the development of treatment resistance highlight the need for patient-derived tumor models that reflect the individual tumors of breast cancer patients and allow a comprehensive analyses and parallel functional validation of individualized and therapeutically targetable vulnerabilities in protein signal transduction pathways. Here, we introduce the generation and application of breast cancer patient-derived 3D microtumors (BC-PDMs). Residual fresh tumor tissue specimens were collected from n = 102 patients diagnosed with breast cancer and subjected to BC-PDM isolation. BC-PDMs retained histopathological characteristics, and extracellular matrix (ECM) components together with key protein signaling pathway signatures of the corresponding primary tumor tissue. Accordingly, BC-PDMs reflect the inter- and intratumoral heterogeneity of breast cancer and its key signal transduction properties. DigiWest®-based protein expression profiling of identified treatment responder and non-responder BC-PDMs enabled the identification of potential resistance and sensitivity markers of individual drug treatments, including markers previously associated with treatment response and yet undescribed proteins. The combination of individualized drug testing with comprehensive protein profiling analyses of BC-PDMs may provide a valuable complement for personalized treatment stratification and response prediction for breast cancer.

Proteomics Approach to Differentiate Protein Extraction Methods in Sugar Beet Leaves.

Interest in alternative plant-based protein sources is continuously growing. Sugar beet leaves have the potential to satisfy that demand due to their high protein content. They are considered as agricultural waste and utilizing them as protein sources can bring them back to the food chain. In this study, isoelectric-point-precipitation, heat-coagulation, ammonium-sulfate precipitation, high-pressure-assisted isoelectric-point precipitation, and high-pressure-assisted heat coagulation methods were used to extract proteins from sugar beet leaves. A mass spectrometry-based proteomic approach was used for comprehensive protein characterization. The analyses yielded 817 proteins, the most comprehensive protein profile on sugar beet leaves to date. Although the total protein contents were comparable, there was a significant difference between the methods for low-abundance proteins. High-pressure-assisted methods showed elevated levels of proteins predominantly located in the chloroplast. Here we showed for the first time that the extraction/precipitation methods may result in different protein profiles that potentially affect the physical and nutritional properties of functional products.

Abstract 6643: Immune checkpoint reporter cell lines based on the protein profiling of ATCC cell lines for cancer immunotherapy drug screening

Abstract The success of immune checkpoint inhibitors in the treatment of various types of cancers and their continued growth in the market have driven burgeoning interests in developing more drugs in this category. However, the intrinsic complexity of the immunological models and the variable drug responses among different cancer types have become the most prominent challenges. To facilitate large scale research projects and drug discovery of immune checkpoint inhibitors, we conducted a comprehensive protein profiling of ATCC’s vast portfolio of human tumor and immune cell lines for several established and novel immune checkpoint molecules. Based on this protein profiling data, we generated immune checkpoint reporter cancer cell lines with high expression of endogenous immune checkpoint molecule ligands (PD-L1, CD155, and B7-H3). The reporter system contains a gamma interferon activation site (GAS)-response element upstream of the luciferase gene, preventing luciferase expression when the immune checkpoint molecule ligand binds to its corresponding receptor that suppresses T cell-mediated antitumor activity. In the presence of a relevant immune checkpoint inhibitor, a luciferase expression-based bioluminescent signal is produced, which can be readily detected and quantitated to evaluate the efficacy, potency, and dynamics of the inhibitor. Our data showed that the bioluminescence in the reporter cancer cells increased approximately 100-250 folds in a dose-dependent manner in response to interferon gamma stimulation, which mimics the signaling from activated CD8+ cytotoxic T cells. The bioluminescence increased approximately 50-100 folds in response to CD8+ primary T cell-conditioned media stimulation. In particular, we observed up to 5-fold increase in luciferase signal from our PD-L1 reporter cell line in response to co-culture with CD8+ primary T cells in the presence of an anti-PD-L1 blocking antibody in a dose-dependent manner. The luciferase expression and endogenous immune checkpoint molecule ligand expression were well maintained after the cell lines had reached &amp;gt;30 population doubling level. These results highlight the robustness and responsiveness of the reporter system for the assessment of T cell-mediated immune responses triggered by checkpoint inhibitors. These immune checkpoint reporter cancer cell lines yield exceptional in vitro and ex vivo assay sensitivity and reproducibility, while simplify the complex immunological model by providing physiologically relevant expression of immune checkpoint molecule ligands, in comparison to similar assays with an artificial checkpoint ligand overexpression system. Citation Format: Hyeyoun Chang, Kevin Tyo, John Foulke, Luping Chen, Zhizhan Gu, Fang Tian. Immune checkpoint reporter cell lines based on the protein profiling of ATCC cell lines for cancer immunotherapy drug screening. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6643.

Egg protein profile and dynamics during embryogenesis in Haemaphysalis flava ticks

Tick eggs contain all essential proteins for embryogenesis, and egg proteins are a potential reservoir of tick-protective antigens. However, the protein profile and dynamics during embryonic development remain unknown. This study aimed to depict the protein profile and dynamics in tick embryogenesis, further providing protein candidates for targeted interventions. Eggs from Haemaphysalis flava ticks were incubated at 28 °C and 85% relative humidity. On days 0 (newly laid eggs without incubation), 7, 14 and 21, eggs were collected, dewaxed and subject to protein extraction. Extracted proteins were digested by filter-aided sample preparation and analyzed by liquid chromatography-tandem mass spectrometry (LC/MS-MS). MS data were searched against an in-house H. flava protein database for tick-derived protein identification. Abundances of 40 selected high-confidence proteins were further quantified by LC-parallel reaction monitoring (PRM)/MS analysis throughout egg incubation. A total of 93 high-confidence proteins were identified in eggs on 0-day incubation. Identified proteins belonged to seven functional categories: transporters, enzymes, proteinase inhibitors, immunity-related proteins, cytoskeletal proteins, heat shock proteins and uncharacterized proteins. The enzyme category contained the most types of proteins. Neutrophil elastase inhibitors represented the most abundant proteins in terms of intensity-based absolute-protein-quantification. LC-PRM/MS revealed that the abundances of 20 proteins increased including enolase, calreticulin, actin, GAPDH et cetera, and the abundances of 11 proteins decreased including vitellogenins, neutrophil elastase inhibitor, carboxypeptidase Q, et cetera from 0- to 21-day incubation. This study provides the most comprehensive egg protein profile and dynamics during tick embryogenesis. Further investigations are needed to test the tick-control efficacy by targeting the egg proteins.

Data-independent acquisition mass spectrometry reveals comprehensive plasma protein profiles in the natural history of patients with hereditary transthyretin amyloidosis (ATTRv)

ABSTRACT Objectives Hereditary transthyretin amyloidosis (ATTRv) is a rare, fatal, autosomal dominant disease with more than 140 mutations discovered. Three phenotypes of amyloid infiltration are neuropathy (ATTRv-PN), cardiopathy (ATTRv-CM), and neuropathy + cardiopathy (ATTRv-MIX). The lack of ATTR-specific biomarkers, difficulties in biopsy evidence, and limited known pathogenic mechanisms have made diagnosis difficult. Newly emerging noninvasive measures for monitoring progression and disease-modifying therapies have improved early diagnosis and patient management. Methods Our research applies the latest technology, Data-Independent Acquisition-Based Quantitative Proteomics (DIA), to reveal comprehensive plasma protein profiles in the natural history of Chinese patients with hereditary transthyretin amyloidosis (ATTRv). We analyzed differentially expressed proteins (DEPs) in three phenotypes (ATTRv-PN, ATTRv-CM, and ATTRv-MIX). Results Serum samples were collected from a total of 18 patients (6 ATTRv-PN, 5 ATTRv-CM, and 7 ATTRv-MIX patients) and 20 healthy participants as a control group. Combined with the results of the proteomic and bioinformatic analyses, we found 30 DEPs and protein interaction networks clustered in KRT family proteins and DSC3 between ATTRv-PN and the control, which were rich in the estrogen signaling pathway and the cell adhesion molecule (CAM) pathway. Conclusion This study demonstrates a global and significant proteomic profile in different stages of ATTRv.

Abstract 14618: Mitochondria-Rich Extracellular Vesicles From Stem Cell-Derived Cardiomyocytes Restore Cardiac Energy Metabolism of Ischemic Myocardium in a Preclinical Pig Model

Introduction: Disruption of mitochondrial energy metabolism underlies the pathophysiology of heart failure. We previously demonstrated that intramyocardial injection of mitochondria-rich extracellular vesicles (M-EVs) from iPSC-derived cardiomyocytes (iCMs) transfer mitochondrial and nonmitochondrial substances into the recipient cardiomyocytes, stimulating mitochondrial biogenesis and functional capacities in the murine acute myocardial infarction (MI) model. However, the therapeutic potential and underlying mechanism in larger animal models remain to be assessed. Methods: M-EVs were isolated from the conditioned medium of iCMs via differential ultracentrifugation. Yorkshire pigs were subjected to myocardial ischemia for one hour via balloon occlusion of the left anterior descending artery. 1.0 x 10 11 M-EVs were injected into the infarct border zone using a percutaneous catheter delivery system (Biocardia, Inc). Cardiac functions were analyzed by cardiac MRI. Liquid chromatography-tandem mass spectrometry was performed to investigate comprehensive protein profiles of M-EV content and pig heart tissue samples. Results: Flow cytometric and electron microscopic analysis revealed that mitochondria inside M-EVs retained membrane potentials and structural integrities. M-EV therapy significantly improved left ventricular (LV) ejection fractions (30.5 vs. 21.2 %, p=0.004) and reduced LV end-diastolic volume (119 vs. 192 mL, p=0.047) at week-4 as compared with PBS control. No difference was observed in the scar size. Proteomics analysis identified a total of 37 significantly enriched proteins in M-EVs. Gene Set Enrichment Analysis of the M-EV proteome revealed significantly upregulated biological processes, including oxidative phosphorylation. STRING network analysis identified enriched proteins that directly or indirectly interact with a proliferator-activated receptor γ coactivator-1 α, a master regulator of mitochondrial biogenesis. Conclusion: M-EV-mediated transfer of mitochondria and enriched proteins enhanced cardiac function by restoring energy metabolism in pig acute MI model. M-EV therapy may represent a novel biologic that directly targets energy metabolism in mitochondria-related diseases.

Proteome Informatics in Tibetan Sheep (Ovis aries) Testes Suggest the Crucial Proteins Related to Development and Functionality.

Testis has an indispensable function in male reproduction of domestic animals. Tibetan sheep (Ovis aries) is a locally adapted breed of sheep raised in the Qinghai-Tibet Plateau, with outsized roles in providing the livelihood for millions of residents. Nevertheless, less is known on how protein expression and their functional roles in developmental testes of such breed limit their use in breeding efforts. In this study, we obtained comprehensive protein profiles from testes of Tibetan sheep at three developmental stages (including pre-puberty, post-puberty, and adulthood) using data-independent acquisition-based proteomic strategy to quantitatively identify the differentially abundant proteins (DAPs) associated with testicular development and function and to unravel the molecular basis of spermatogenesis. A total of 6,221 proteins were differentially expressed in an age-dependent manner. The reliability of the gene expression abundance was corroborated by quantitative PCR and targeted parallel reaction monitoring. These DAPs were significantly enriched to biological processes concerning spermatid development and sperm deformation, mitosis, glycolytic process, cell-cell/extracellular matrix (ECM) junctions, cell proliferation, apoptosis, and migration and to the pathways including, developmental process and sexual reproduction-related (such as VEGF, estrogen, insulin, GnRH, Hippo, PI3K-Akt, mTOR, MAPK, and AMPK), and testicular cell events-related pathways (such as tight/gap/adherens junctions, ECM-receptor interaction, regulation of actin cytoskeleton, glycolysis, cell cycle, and meiosis). Based on these bioinformatics analysis, we constructed four protein–protein interaction network, among which the proteins are involved in mitosis, meiosis, spermiogenesis, and testicular microenvironment, respectively. Altogether, these bioinformatics-based sequencing results suggest that many protein-coding genes were expressed in a development-dependent manner in Tibetan sheep testes to contribute to the testicular cell development and their surrounding microenvironment remodeling at various stages of spermatogenesis. These findings have important implications for further understanding of the mechanisms underlying spermatogenesis in sheep and even other plateau-adapted animals.

Abstract 3801: Comprehensive characterization of the biology of inflammatory bowel disease-associated colorectal cancer using the GeoMx® Digital Spatial Profiler (DSP)

Abstract Inflammatory bowel diseases (IBD) ulcerative colitis (UC) and Crohn's disease (CD) significantly increases the risk for development of colorectal cancer (CRC). However, the exact molecular mechanisms that underlie the transition from inflammation to cancer are not fully understood, and a systematic characterization of the immunopathogenesis of IBD-associated CRC is lacking. To address this issue, we will perform a comprehensive and spatial tissue analysis using the Cancer Transcriptome Atlas (CTA) on the NanoString GeoMx® Digital Spatial Profiler (DSP). The CTA panel is designed to profile the global immune response and all aspects of tumor microenvironment biology, including the 18-gene Tumor Inflammation Signature (TIS). For DSP analysis, a total of 20 FFPE samples (5 colon cancer patients, 5 UC patients, 5 CD patients, and 5 matched normal samples will be spatially profiled for up to 1,800 genes included on the CTA. Selection of regions of interest (ROI) will be guided by H&amp;E staining and fluorescent markers (CD45, PanCK, Syto13), and profiling of tumor and TME regions will be achieved through segmenting by PanCK+/PanCK- followed by collection of indexed oligonucleotides and sequenced on NextSeq 550 Illumina instrument. NGS reads will be processed into digital counts, normalized and mapped back to each ROI, generating a map of transcript activity within each ROI. For all samples crypt and villus regions will be selected for a detailed spatial analysis. Subsequently, using the proprietary multiplex immunofluorescence (mIF) platform MultiOmyx™ we will characterize the protein immune cell landscape in IBD-associated CRC, using a custom immune panel applied to adjacent slides. With this study we hope to highlight genes and pathways involved in the transition from inflammation to cancer by combining large-scale spatial gene profiling by DSP CTA with a comprehensive spatial protein profiling by MultiOmyx. It is our hope that this will provide some much-needed understanding of the underlying mechanisms leading from an IBD disorder to the development of colon cancer. Citation Format: Anna Juncker-Jensen, Lakshmi Chandramohan. Comprehensive characterization of the biology of inflammatory bowel disease-associated colorectal cancer using the GeoMx® Digital Spatial Profiler (DSP) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3801.

An omic and multidimensional spatial atlas from serial biopsies of an evolving metastatic breast cancer

SummaryMechanisms of therapeutic resistance and vulnerability evolve in metastatic cancers as tumor cells and extrinsic microenvironmental influences change during treatment. To support the development of methods for identifying these mechanisms in individual people, here we present an omic and multidimensional spatial (OMS) atlas generated from four serial biopsies of an individual with metastatic breast cancer during 3.5 years of therapy. This resource links detailed, longitudinal clinical metadata that includes treatment times and doses, anatomic imaging, and blood-based response measurements to clinical and exploratory analyses, which includes comprehensive DNA, RNA, and protein profiles; images of multiplexed immunostaining; and 2- and 3-dimensional scanning electron micrographs. These data report aspects of heterogeneity and evolution of the cancer genome, signaling pathways, immune microenvironment, cellular composition and organization, and ultrastructure. We present illustrative examples of how integrative analyses of these data reveal potential mechanisms of response and resistance and suggest novel therapeutic vulnerabilities.

Investigation of cattle plasma proteome in response to pain and inflammation using next generation proteomics technique, SWATH-MS.

Pain assessment in farm animals has primarily relied on a combination of behavioral and physiological responses, although these are relatively subjective and difficult to quantify. It is essential to develop more effective biomarkers of pain in production animals since they are frequently exposed to routine surgical husbandry procedures. More effective biomarkers of pain would improve welfare, limit the loss of productivity associated with pain and permit better assessment of analgesics. This study aimed to investigate the use of a modern mass spectrometry data independent acquisition strategy, termed Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS), to detect candidate protein biomarkers that are known to associate with nociceptive and inflammatory processes in cattle, which could then be used to assess the efficacy of potential analgesics. Calves were randomly divided into two groups that were either surgically dehorned or subjected to restraint stress, without provision of anaesthesia or analgesia in accordance with current industry standards. Samples were analysed before and after dehorning at multiple timepoints. Significant changes in protein concentrations were detected predominantly at 24 and 96 h following dehorning, including kininogens, proteins associated with the coagulation and complement cascades and serine protease inhibitors. Gene ontology analysis revealed that the identified candidate biomarkers were associated with stress, wound healing, immune response, blood coagulation and the inflammatory and acute phase responses, which could be expected following surgical damage to tissues, but can now be more objectively assessed. These results offer more definitive and quantitative monitoring of response to tissue injury induced pain and inflammation.

Organ-Based Proteome and Post-Translational Modification Profiling of a Widely Cultivated Tropical Water Fish, Labeo rohita.

Proteomics has enormous applications in human and animal research. However, proteomic studies in fisheries science are quite scanty particularly for economically important species. Few proteomic studies have been carried out in model fish species, but comprehensive proteomics of aquaculture species are still scarce. This study aimed to perform a comprehensive organ-based protein profiling of important tissue samples for one of the most important aquaculture species,Labeo rohita.Deep proteomic profiling of 17 histologically normal tissues, blood plasma, and embryo provided mass-spectrometric evidence for 8498 proteins at 1% false discovery rate that make up about 26% of the total annotated protein-coding sequences in Rohu. Tissue-wise expression analysis was performed, and the presence of several biologically important proteins was also verified using a targeted proteomic approach. We identified the global post-translational modifications (PTMs) in terms of acetylation (N-terminus and lysine), methylation (N-terminus, lysine, and arginine), and phosphorylation (serine, threonine, and tyrosine) to present a comprehensive proteome resource. An interactive web-based portal has been developed for an overall landscape of protein expression across the studied tissues of Labeo rohita (www.fishprot.org). This draft proteome map of Labeo rohita would advance basic and applied research in aquaculture to meet the most critical challenge of providing food and nutritional security to an increasing world population.

DIA-Based Quantitative Proteomics Reveals the Protein Regulatory Networks of Floral Thermogenesis in Nelumbo nucifera.

The sacred lotus (Nelumbo nucifera) can maintain a stable floral chamber temperature between 30 and 35 °C when blooming despite fluctuations in ambient temperatures between about 8 and 45 °C, but the regulatory mechanism of floral thermogenesis remains unclear. Here, we obtained comprehensive protein profiles from receptacle tissue at five developmental stages using data-independent acquisition (DIA)-based quantitative proteomics technology to reveal the molecular basis of floral thermogenesis of N. nucifera. A total of 6913 proteins were identified and quantified, of which 3513 differentially abundant proteins (DAPs) were screened. Among them, 640 highly abundant proteins during the thermogenic stages were mainly involved in carbon metabolism processes such as the tricarboxylic acid (TCA) cycle. Citrate synthase was identified as the most connected protein in the protein-protein interaction (PPI) network. Next, the content of alternative oxidase (AOX) and plant uncoupling protein (pUCP) in different tissues indicated that AOX was specifically abundant in the receptacles. Subsequently, a protein module highly related to the thermogenic phenotype was identified by the weighted gene co-expression network analysis (WGCNA). In summary, the regulation mechanism of floral thermogenesis in N. nucifera involves complex regulatory networks, including TCA cycle metabolism, starch and sucrose metabolism, fatty acid degradation, and ubiquinone synthesis, etc.

Longitudinal plasma protein profiling of newly diagnosed type 2 diabetes.

BackgroundComprehensive proteomics profiling may offer new insights into the dysregulated metabolic milieu of type 2 diabetes, and in the future, serve as a useful tool for personalized medicine. This calls for a better understanding of circulating protein patterns at the early stage of type 2 diabetes as well as the dynamics of protein patterns during changes in metabolic status.MethodsTo elucidate the systemic alterations in early-stage diabetes and to investigate the effects on the proteome during metabolic improvement, we measured 974 circulating proteins in 52 newly diagnosed, treatment-naïve type 2 diabetes subjects at baseline and after 1 and 3 months of guideline-based diabetes treatment, while comparing their protein profiles to that of 94 subjects without diabetes.FindingsEarly stage type 2 diabetes was associated with distinct protein patterns, reflecting key metabolic syndrome features including insulin resistance, adiposity, hyperglycemia and liver steatosis. The protein profiles at baseline were attenuated during guideline-based diabetes treatment and several plasma proteins associated with metformin medication independently of metabolic variables, such as circulating EPCAM.InterpretationThe results advance our knowledge about the biochemical manifestations of type 2 diabetes and suggest that comprehensive protein profiling may serve as a useful tool for metabolic phenotyping and for elucidating the biological effects of diabetes treatments.FundingThis work was supported by the Swedish Heart and Lung Foundation, the Swedish Research Council, the Erling Persson Foundation, the Knut and Alice Wallenberg Foundation, and the Swedish state under the agreement between the Swedish government and the county councils (ALF-agreement).

Characterization of Local and Systemic Impact of Whitefly (Bemisia tabaci) Feeding and Whitefly-Transmitted Tomato Mottle Virus Infection on Tomato Leaves by Comprehensive Proteomics

Tomato mottle virus (ToMoV) is a single-stranded DNA (ssDNA) begomovirus transmitted to solanaceous crops by the whitefly species complex (Bemisia tabaci), causing stunted growth, leaf mottling, and reduced yield. Using a genetic repertoire of seven genes, ToMoV pathogenesis includes the manipulation of multiple plant biological processes to circumvent antiviral defenses. To further understand the effects of whitefly feeding and whitefly-transmitted ToMoV infection on tomato plants (Solanum lycopersicum ‘Florida Lanai’), we generated comprehensive protein profiles of leaves subjected to feeding by either viruliferous whiteflies harboring ToMoV, or non-viruliferous whiteflies, or a no-feeding control. The effects of whitefly feeding and ToMoV infection were measured both locally and systemically by sampling either a mature leaf directly from the site of clip-cage confined whitefly feeding, or from a newly formed leaf 10 days post feeding (dpf). At 3 dpf, tomato’s response to ToMoV included proteins associated with translation initiation and elongation as well as plasmodesmata dynamics. In contrast, systemic impacts of ToMoV on younger leaves 10 dpf were more pronounced and included a virus-specific change in plant proteins associated with mRNA maturation and export, RNA-dependent DNA methylation, and other antiviral plant processes. Our analysis supports previous findings and provides novel insight into tomato’s local and systemic response to whitefly feeding and ToMoV infection.

Single-cell proteomic analysis.

The ability to comprehensively profile proteins in every individual cell of complex biological systems is crucial to advance our understanding of normal physiology and disease pathogenesis. Conventional bulk cell experiments mask the cell heterogeneity in the population, while the single-cell imaging methods suffer from the limited multiplexing capacities. Recent advances in microchip-, mass spectrometry-, and reiterative staining-based technologies have enabled comprehensive protein profiling in single cells. These approaches will bring new insights into a variety of biological and biomedical fields, such as signaling network regulation, cell heterogeneity, tissue architecture, disease diagnosis, and treatment monitoring. In this article, we will review the recent advances in the development of single-cell proteomic technologies, describe their advantages, discuss the current limitations and challenges, and propose potential solutions. We will also highlight the wide applications of these technologies in biology and medicine. This article is categorized under: Cancer > Molecular and Cellular Physiology.

Infinity Flow: comprehensive single-cell protein profiling via massively parallel flow cytometry and machine learning

Abstract Modern flow cytometers can quantify around 20 proteins at the single-cell level. Higher-dimensional alternatives exist, such as mass cytometry, or oligonucleotides-tagged antibodies - but they feature lower cellular throughput, higher cost and are not as widely available. To overcome these limitations, we propose a pipeline which allows the quantification of hundreds of proteins across millions of single cells. Experimentally, cells are stained with a customizable 10–20 “backbone” antibody panel which defines the cellular population structure of the sample. This is followed by aliquoting into hundreds (200–300) of individual wells, each containing a unique “exploratory” antibody. Data acquisition is performed using conventional flow cytometry. Computationally, multivariate non-linear regression models are trained, one for each well. These models learn how to impute an exploratory antibody signal from backbone measurements. The models are each applied to the backbone data to obtain hundreds of imputed measurements across millions of single cells. We applied this workflow cell suspensions from the lungs of C57/B6 mice, resulting in 2,660,000 events with imputed expression levels for 266 proteins. Imputed expression and co-expression patterns are accurate and consensual across regression models, allowing near-exhaustive annotation of both immune and non-immune cells, and finer granularity than dimensionality reduction or clustering. By enabling broad characterization of cellular networks at the protein level, this pipeline is well-suited for systems immunology approaches. Given the wealth of data generated by this pipeline and its accessibility, we anticipate it will be widely adopted.