Proteomics Research Research Articles

Facile synthesis of titanium-rich carbonaceous material via Schiff base reaction for efficient separation and analysis of phosphorylated peptides and exosomes from human serum

Efficient capture of phosphorylated peptides and phosphorylated exosomes is important for in-depth proteomics research. In this work, a carbonaceous material (denoted as CTs@DHA) was synthesized based on the Schiff base reaction by hydrothermal carbonization using chitosan (denoted as CTs) and 2,3-dihydroxy terephthalaldehyde (denoted as DHA) as ligands and water as the sole component of the solvent. The most important thing is that the experimental process is simple, environmentally friendly, and time-saving. Based on the immobilized metal affinity chromatography (IMAC) strategy, titanium ions were chelated with CTs@DHA to obtain a novel material (denoted as CTs@DHA@Ti4+) to capture exosomes and phosphopeptides. The CTs@DHA@Ti4+ captured phosphopeptides with high selectivity (the molar ratio of BSA to β-casein was 2000:1) and satisfactory sensitivity (2 fmol), with excellent loading capacity (83.3 μg/mg). Additionally, this material was successfully applied in complex bio-sample analysis; four endogenous phosphopeptides from the serum of uremic patients, 16 phosphopeptides from human saliva, and exosomes from human serum were detected. After being analyzed with nano-LC MS/MS, 12 phosphopeptides with 10 phosphorylation sites from 10 phosphoproteins were detected in normal serum, and 14 phosphopeptides with 16 phosphorylation sites from 13 phosphoproteins were detected in the serum of uremic patients. All experiments have shown that this material provides an effective method for isolating exosomes and enriching phosphopeptides, and it has bright application prospects in disease research and proteomics research.

Precise Control of Trypsin Immobilization by a Programmable DNA Tetrahedron Designed for Ultrafast Proteome Digestion and Accurate Protein Quantification.

In proteomics research, with advantages including short digestion times and reusable applications, immobilized enzyme reactors (IMERs) have been paid increasing attention. However, traditional IMERs ignore the reasonable spatial arrangement of trypsin on the supporting matrixes, resulting in the partial overlapping of the active domain on trypsin and reducing digesting efficiency. In this work, a DNA tetrahedron (DNA TET)-based IMER Fe3O4-GO-AuNPs-DNA TET-Trypsin was designed and prepared. The distance between vertices of DNA TETs effectively controls the distribution of trypsin on the nanomaterials; thus, highly efficient protein digestion and accurate quantitative results can be achieved. Compared to the in-solution digestion (12-16 h), the sequence coverage of bovine serum albumin was up to 91% after a 2-min digestion by the new IMER. In addition, 3328 proteins and 18,488 peptides can be identified from HeLa cell protein extract after a 20-min digestion. For the first time, human growth hormone reference material was rapidly and accurately quantified after a 4-h digestion by IMER. Therefore, this new IMER has great application potential in proteomics research and SI traceable quantification.

Proteomic meta-study harmonization, mechanotyping and drug repurposing candidate prediction with ProHarMeD

Proteomics technologies, which include a diverse range of approaches such as mass spectrometry-based, array-based, and others, are key technologies for the identification of biomarkers and disease mechanisms, referred to as mechanotyping. Despite over 15,000 published studies in 2022 alone, leveraging publicly available proteomics data for biomarker identification, mechanotyping and drug target identification is not readily possible. Proteomic data addressing similar biological/biomedical questions are made available by multiple research groups in different locations using different model organisms. Furthermore, not only various organisms are employed but different assay systems, such as in vitro and in vivo systems, are used. Finally, even though proteomics data are deposited in public databases, such as ProteomeXchange, they are provided at different levels of detail. Thus, data integration is hampered by non-harmonized usage of identifiers when reviewing the literature or performing meta-analyses to consolidate existing publications into a joint picture. To address this problem, we present ProHarMeD, a tool for harmonizing and comparing proteomics data gathered in multiple studies and for the extraction of disease mechanisms and putative drug repurposing candidates. It is available as a website, Python library and R package. ProHarMeD facilitates ID and name conversions between protein and gene levels, or organisms via ortholog mapping, and provides detailed logs on the loss and gain of IDs after each step. The web tool further determines IDs shared by different studies, proposes potential disease mechanisms as well as drug repurposing candidates automatically, and visualizes these results interactively. We apply ProHarMeD to a set of four studies on bone regeneration. First, we demonstrate the benefit of ID harmonization which increases the number of shared genes between studies by 50%. Second, we identify a potential disease mechanism, with five corresponding drug targets, and the top 20 putative drug repurposing candidates, of which Fondaparinux, the candidate with the highest score, and multiple others are known to have an impact on bone regeneration. Hence, ProHarMeD allows users to harmonize multi-centric proteomics research data in meta-analyses, evaluates the success of the ID conversions and remappings, and finally, it closes the gaps between proteomics, disease mechanism mining and drug repurposing. It is publicly available at https://apps.cosy.bio/proharmed/.

Proteomic research and protein biochemistry for targeted therapy of anti-cancer drugs

Proteomic research and protein biochemistry for targeted therapy of anti-cancer drugs

Proteomic Analysis Reveals Potential Exosomal Biomarkers in Patients With Sporadic Alzheimer Disease.

Despite substantial progress made in the past decades, the pathogenesis of sporadic Alzheimer disease (sAD) and related biological markers of the disease are still controversially discussed. Cerebrospinal fluid and functional brain imaging markers have been established to support the clinical diagnosis of sAD. Yet, due to the invasiveness of such diagnostics, less burdensome markers have been increasingly investigated in the past years. Among such markers, extracellular vesicles may yield promise in (early) diagnostics and treatment monitoring in sAD. In this pilot study, we collected the blood plasma of 18 patients with sAD and compared the proteome of extracted extracellular vesicles with the proteome of 11 age-matched healthy controls. The resulting proteomes were characterized by Gene Ontology terms and between-group statistics. Ten distinct proteins were found to significantly differ between sAD patients and controls (P<0.05, False Discovery Rate, corrected). These proteins included distinct immunoglobulins, fibronectin, and apolipoproteins. These findings lend further support for exosomal changes in neurodegenerative disorders, and particularly in sAD. Further proteomic research could decisively advance our knowledge of sAD pathophysiology as much as it could foster the development of clinically meaningful biomarkers.

MassSpecPreppy-An end-to-end solution for automated protein concentration determination and flexible sample digestion for proteomics applications.

In proteomics, fast, efficient, and highly reproducible sample preparation is of utmost importance, particularly in view of fast scanning mass spectrometers enabling analyses of large sample series. To address this need, we have developed the web application MassSpecPreppy that operates on the open science OT-2 liquid handling robot from Opentrons. This platform can prepare up to 96 samples at once, performing tasks like BCA protein concentration determination, sample digestion with normalization, reduction/alkylation and peptide elution into vials or loading specified peptide amounts onto Evotips in an automated and flexible manner. The performance of the developed workflows using MassSpecPreppy was compared with standard manual sample preparation workflows. The BCA assay experiments revealed an average recovery of 101.3% (SD:±7.82%) for the MassSpecPreppy workflow, while the manual workflow had a recovery of 96.3% (SD:±9.73%). The species mix used in the evaluation experiments showed that 94.5% of protein groups for OT-2 digestion and 95% for manual digestion passed the significance thresholds with comparable peptide level coefficient of variations. These results demonstrate that MassSpecPreppy is a versatile and scalable platform for automated sample preparation, producing injection-ready samples for proteomics research.

Mitochondrial proteome research: the road ahead.

Mitochondria are multifaceted organelles with key roles in anabolic and catabolic metabolism, bioenergetics, cellular signalling and nutrient sensing, and programmed cell death processes. Their diverse functions are enabled by a sophisticated set of protein components encoded by the nuclear and mitochondrial genomes. The extent and complexity of the mitochondrial proteome remained unclear for decades. This began to change 20 years ago when, driven by the emergence of mass spectrometry-based proteomics, the first draft mitochondrial proteomes were established. In the ensuing decades, further technological and computational advances helpedto refine these 'maps', with current estimates of the core mammalian mitochondrial proteome ranging from 1,000 to 1,500 proteins. The creation of these compendia provided a systemic view of an organelle previously studied primarily in a reductionist fashion and has accelerated both basic scientific discovery and the diagnosis and treatment of human disease. Yet numerous challenges remain in understanding mitochondrial biology and translating this knowledge into the medical context. In this Roadmap, we propose a path forward for refining the mitochondrial protein map to enhance its discovery and therapeutic potential. We discuss how emerging technologies can assist the detection of new mitochondrial proteins, reveal their patterns of expression across diverse tissues and cell types, and provide key information on proteoforms. We highlight the power of an enhanced map for systematically defining the functions of its members. Finally, we examine the utility of an expanded, functionally annotated mitochondrial proteome in a translational setting for aiding both diagnosis of mitochondrial disease and targeting of mitochondria for treatment.

Identification of the Kupffer cell-derived circulating IGFBP-3 as a universal radiation biomarker for heavy ion, proton, and X-ray exposure

The minimally invasive biomarkers that can facilitate a rapid dose assessment are valuable for the early medical treatment when accidental or occupational radiation exposure happens. Our previous proteomic research identified one kind of circulating protein, Insulin-like Growth Factor Binding Protein 3 (IGFBP-3), which showed a significant increase after total body exposure of mice to carbon ions and X-rays. However, several critical issues such as the responses to diverse radiation, the origin and underlying mechanism in radiation response obstruct the utilization of circulating IGFBP-3 as a reliable radiation biomarker. In this study, mice were subjected to total or partial body irradiation with carbon ions, protons or X-rays, or treated with chloroform as a comparison. The level of IGFBP-3 in serum and different organs were measured via Enzyme Linked Immunosorbent Assay (ELISA), Western blot (WB) and Immunohistochemistry (IHC). A significant increase of IGFBP-3 was discovered in serum and liver tissue post-irradiation with three kinds of radiation, but absent when challenged with chloroform. Likewise, a similar response was also observed in blood samples from patients receiving radiotherapy. Moreover, the effect of radiation on three main hepatic cells was investigated, the findings indicated that IGFBP-3 could be detected in the culture medium of Kupffer cells (MKC) alone and was elevated in cells and cultured medium of MKC post-irradiation. Additionally, we observed a co-expression effect between P53 and IGFBP-3 in liver tissues and MKC post-irradiation. Along with down-regulation of Trp53 by siRNA, the response of IGFBP-3 to radiation was attenuated. The present study demonstrated that circulating IGFBP-3 could be a promising universal biomarker for complex environmental radiation exposure, and the upregulation of IGFBP-3 is attributed to the MKC in a P53-dependent manner. Circulating IGFBP-3 assays would offer rapid, convenient and effective dose and toxicity assessment methods in occupational exposure or radiation disaster management.

Spectral library search for improved TMTpro labelled peptide assignment in human plasma proteomics.

Clinical biomarker discovery is often based on the analysis of human plasma samples. However, the high dynamic range and complexity of plasma pose significant challenges to mass spectrometry-based proteomics. Current methods for improving protein identifications require laborious pre-analytical sample preparation. In this study, we developed and evaluated a TMTpro-specific spectral library for improved protein identification in human plasma proteomics. The library was constructed by LC-MS/MS analysis of highly fractionated TMTpro-tagged human plasma, human cell lysates, and relevant arterial tissues. The library was curated using several quality filters to ensure reliable peptide identifications. Our results show that spectral library searching using the TMTpro spectral library improves the identification of proteins in plasma samples compared to conventional sequence database searching. Protein identifications made by the spectral library search engine demonstrated a high degree of complementarity with the sequence database search engine, indicating the feasibility of increasing the number of protein identifications without additional pre-analytical sample preparation. The TMTpro-specific spectral library provides a resource for future plasma proteomics research and optimization of search algorithms for greater accuracy and speed in protein identifications in human plasma proteomics, and is made publicly available to the research community via ProteomeXchange with identifier PXD042546.

Plasma proteins as potential biomarkers of aging of single tissue and cell type.

Plasma proteins serve as biomarkers of aging and various age-related diseases. While a number of plasma proteins have been identified that increase or decrease with age, the interpretation of each protein is challenging. This is due to the nature of plasma, which is a mixture of factors secreted by many different tissues and cells. Therefore, the catalog of age-related proteins secreted by a single cell type in a single tissue would be useful for understanding tissue-specific aging patterns. In this study, the author addressed this challenge by integrative data mining of the Human Protein Atlas and the recently published result of large-scale aging proteomics research. Finally, we identified the 17 age-related proteins produced by a single tissue and a single cell type: MBL2 and HP in the liver (hepatocytes), SFTPC in the lung (type II alveolar cells), PRL and POMC in the pituitary (anterior cells), GCG, CUZD1 and CPA2 in the pancreas (pancreatic cells), MYBPC1 in skeletal muscle (myocytes), PTH in the parathyroid gland (glandular cells), LPO and AMY1A in the salivary gland (glandular cells), INSL3 in the male testis (Leydig cells), KLK3 and KLK4 in the male prostate (glandular cells), MPO and ACP5 in immune cells. This list of proteins would be potentially useful for understanding age-related changes in the plasma proteome and inter-tissue networks.

Application of Proteomics in Alzheimer’s Disease: A Mini Review

Alzheimer’s disease (AD) is classified as one of neurodegenerative disease caused by neuronal death. It is characterized as memory impairment, including the inability to produce new memories. Since AD has low treatment effectiveness, proteomics research opens possibilities for advancement. Proteomics is the study of proteomes produced by the disease-bearing host to identify and understand diseases. In this case, to investigate the use of protein as a reliable molecular entity and their involvement in AD. Therefore, this review focused on three main applications of proteomics; the potential use of proteomics as a diagnostic tool for AD, the use of proteomics to assess the treatment progression of AD and the advancement in AD research. The review discussed three research areas utilizing the proteomics approach: ageing, behavioural, and demographic research of AD populations. Proteomic approaches have also been shown to be effective to discover the biomarkers for infectious diseases, cancers, heart diseases, and neurological disorders. Although much work remained to be done, the proteomics approach is an interesting method to be carried out in detecting AD at an earlier stage and will be very useful for AD treatment and management in the future.

A Multipathway Phosphopeptide Standard for Rapid Phosphoproteomics Assay Development

Recent advances in methodology have made phosphopeptide analysis a tractable problem for many proteomics researchers. There are now a wide variety of robust and accessible enrichment strategies to generate phosphoproteomes while free or inexpensive software tools for quantitation and site localization have simplified phosphoproteome analysis workflow tremendously. As a research group under the Association for Biomolecular Resource Facilities umbrella, the Proteomics Standards Research Group has worked to develop a multipathway phosphopeptide standard based on a mixture of heavy-labeled phosphopeptides designed to enable researchers to rapidly develop assays. This mixture contains 131 mass spectrometry vetted phosphopeptides specifically chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling networks: AMPK signaling, death and apoptosis signaling, ErbB signaling, insulin/insulin-like growth factor-1 signaling, mTOR signaling, PI3K/AKT signaling, and stress (p38/SAPK/JNK) signaling. Here, we describe a characterization of this mixture spiked into a HeLa tryptic digest stimulated with both epidermal growth factor and insulin-like growth factor-1 to activate the MAPK and PI3K/AKT/mTOR pathways. We further demonstrate a comparison of phosphoproteomic profiling of HeLa performed independently in five labs using this phosphopeptide mixture with data-independent acquisition. Despite different experimental and instrumentation processes, we found that labs could produce reproducible, harmonized datasets by reporting measurements as ratios to the standard, while intensity measurements showed lower consistency between labs even after normalization. Our results suggest that widely available, biologically relevant phosphopeptide standards can act as a quantitative "yardstick" across laboratories and sample preparations enabling experimental designs larger than a single laboratory can perform. Raw data files are publicly available in the MassIVE dataset MSV000090564.

Proteomics Research Reveals the Molecular Mechanism by Which Grape Seed Oil Inhibits Tuber Sprouting in Potato

Potato tubers are rich in starch, vitamins, protein, minerals, and other nutrients. However, tuber sprouting produces solanine and reduces the commodity value of potatoes during storage. At present, it is known that some plant essential oils can inhibit tuber sprouting. It has been reported that grape seed oil (GSO) has antioxidant, anti-inflammatory, and anticancer characteristics, reducing blood lipids and delaying aging. In this study, we found for the first time that GSO delayed tuber sprouting, and the soluble sugar content and peroxidase activity changed after 60 days of GSO treatment. Furthermore, a comparative proteomic analysis of tuber bud eyes showed that after 30 days of GSO treatment, there were 206 and 129 differentially abundant proteins (DAPs) with increased and decreased abundance levels, respectively. After analysis, we found that 15 ROS-related proteins and 14 proteins involved in energy metabolism were DAPs. Among them, gamma aminobutyrate transaminase 1 had decreased abundance after GSO treatment. Meanwhile, the transcription level of genes related to GABA synthesis increased significantly according to qRT-PCR analysis. Our results provide new approaches to the proteomic mechanism of potato sprouting after GSO treatment and provide a theoretical basis for the application of GSO in inhibiting potato seed sprouts.

AlphaPeptStats: an open-source Python package for automated and scalable statistical analysis of mass spectrometry-based proteomics.

The widespread application of mass spectrometry (MS)-based proteomics in biomedical research increasingly requires robust, transparent, and streamlined solutions to extract statistically reliable insights. We have designed and implemented AlphaPeptStats, an inclusive Python package with currently with broad functionalities for normalization, imputation, visualization, and statistical analysis of label-free proteomics data. It modularly builds on the established stack of Python scientific libraries and is accompanied by a rigorous testing framework with 98% test coverage. It imports the output of a range of popular search engines. Data can be filtered and normalized according to user specifications. At its heart, AlphaPeptStats provides a wide range of robust statistical algorithms such as t-tests, analysis of variance, principal component analysis, hierarchical clustering, and multiple covariate analysis-all in an automatable manner. Data visualization capabilities include heat maps, volcano plots, and scatter plots in publication-ready format. AlphaPeptStats advances proteomic research through its robust tools that enable researchers to manually or automatically explore complex datasets to identify interesting patterns and outliers. AlphaPeptStats is implemented in Python and part of the AlphaPept framework. It is released under a permissive Apache license. The source code and one-click installers are freely available and on GitHub athttps://github.com/MannLabs/alphapeptstats.

Systematic Proteomics Study on the Embryonic Development of Danio rerio.

The early development of zebrafish (Danio rerio) is a complex and dynamic physiological process involving cell division, differentiation, and movement. Currently, the genome and transcriptome techniques have been widely used to study the embryonic development of zebrafish. However, the research of proteomics based on proteins that directly execute functions is relatively vacant. In this work, we apply label-free quantitative proteomics to explore protein profiling during zebrafish's embryogenesis, and a total of 5961 proteins were identified at 10 stages of zebrafish's early development. The identified proteins were divided into 11 modules according to weighted gene coexpression network analysis (WGCNA), and the characteristics between modules were significantly different. For example, mitochondria-related functions enriched the early development of zebrafish. Primordial germ cell-related proteins were identified at the 4-cell stage, while the eye development event is dominated at 5 days post fertilization (dpf). By combining with published transcriptomics data, we discovered some proteins that may be involved in activating zygotic genes. Meanwhile, 137 novel proteins were identified. This study comprehensively analyzed the dynamic processes in the embryonic development of zebrafish from the perspective of proteomics. It provided solid data support for further understanding of the molecular mechanism of its development.

In-situ PLL-g-PEG Functionalized Nanopore for Enhancing Protein Characterization.

Single-molecule nanopore detection technology has revolutionized proteomics research by enabling highly sensitive and label-free detection of individual proteins. Herein, we designed a small, portable, and leak-free flowcell made of PMMA for nanopore experiments. In addition, we developed an in situ functionalizing PLL-g-PEG approach to produce non-sticky nanopores for measuring the volume of diseases-relevant biomarker, such as the Alpha-1 antitrypsin (AAT) protein. The in situ functionalization method allows continuous monitoring, ensuring adequate functionalization, which can be directly used for translocation experiments. The functionalized nanopores exhibit improved characteristics, including an increased nanopore lifetime and enhanced translocation events of the AAT proteins. Furthermore, we demonstrated the reduction in the translocation event's dwell time, along with an increase in current blockade amplitudes and translocation numbers under different voltage stimuli. The study also successfully measures the single AAT protein volume (253 nm3 ), which closely aligns with the previously reported hydrodynamic volume. The real-time in situ PLL-g-PEG functionalizing method and the developed nanopore flowcell hold great promise for various nanopores applications involving non-sticky single-molecule characterization.

Dominant sanitary significant microflora of meat

We conducted bacteriological study of samples of small-sized beef, as well as minced pork, beef, homemade, purchased in retail chains. Bacteriological, bacterioscopic and proteomic research methods were used. The total bacterial contamination, freshness indicators, the presence of pathogens of bacterial diseases, opportunistic microorganisms and representatives of the dominant saprophytic microflora were determined. The absence of Salmonella, listeria, Pseudomonas aeruginosa, Staphylococcus aureus, Proteus, yeast and molds was established. The dominant microflora of meat are Enterobacteria of the genus Citrobacter, Entrrococcus faecium.

A multi-omics dataset of human transcriptome and proteome stable reference

The development of high-throughput omics technology has greatly promoted the development of biomedicine. However, the poor reproducibility of omics techniques limits their application. It is necessary to use standard reference materials of complex RNAs or proteins to test and calibrate the accuracy and reproducibility of omics workflows. The transcriptome and proteome of most cell lines shift during culturing, which limits their applicability as standard samples. In this study, we demonstrated that the human hepatocellular cell line MHCC97H has a very stable transcriptome (r = 0.983~0.997) and proteome (r = 0.966~0.988 for data-dependent acquisition, r = 0.970~0.994 for data-independent acquisition) after 9 subculturing generations, which allows this steady standard sample to be consistently produced on an industrial scale in long term. Moreover, this stability was maintained across labs and platforms. In sum, our study provides omics standard reference material and reference datasets for transcriptomic and proteomics research. This helps to further standardize the workflow and data quality of omics techniques and thus promotes the application of omics technology in precision medicine.

The synthesis and functionalization of metal organic frameworks and their applications for the selective separation of proteins/peptides.

Recently, proteins separation has drawn great interest for the full investigation of a proteome because the proteins separation is the precondition when conducting clinical research or proteomics research. Metal organic frameworks (MOFs) are fabricated via covalent connection between organic ligands and metal ions/clusters units. MOFs have attracted much attention due to the ultra-high specific surface area, tunable structure, more metal site or unsaturated site, and chemical stability. Over the past decade, different functionalization types of MOFs have been reported in combination with amino acids, nucleic acids, proteins, polymers, and nanoparticles for various applications. In this review, the synthesis and functionalization of MOFs have been thoroughly discussed, and we introduced the existing problems and development trends in these fields. Furthermore, MOFs as advanced adsorbents for selective separation of proteins/peptides are summarized. Additionally, we present a comprehensive prospects and challenges in the preparation of robust functional MOFs-based adsorbents and make a final outlook on their future development prospects in selective separation of proteins/peptides.

Proteomic Alteration in the Progression of Multiple Myeloma: A Comprehensive Review.

Multiple myeloma (MM) is an incurable hematologic malignancy. Most MM patients are diagnosed at a late stage because the early symptoms of the disease can be uncertain and nonspecific, often resembling other, more common conditions. Additionally, MM patients are commonly associated with rapid relapse and an inevitable refractory phase. MM is characterized by the abnormal proliferation of monoclonal plasma cells in the bone marrow. During the progression of MM, massive genomic alterations occur that target multiple signaling pathways and are accompanied by a multistep process involving differentiation, proliferation, and invasion. Moreover, the transformation of healthy plasma cell biology into genetically heterogeneous MM clones is driven by a variety of post-translational protein modifications (PTMs), which has complicated the discovery of effective treatments. PTMs have been identified as the most promising candidates for biomarker detection, and further research has been recommended to develop promising surrogate markers. Proteomics research has begun in MM, and a comprehensive literature review is available. However, proteomics applications in MM have yet to make significant progress. Exploration of proteomic alterations in MM is worthwhile to improve understanding of the pathophysiology of MM and to search for new treatment targets. Proteomics studies using mass spectrometry (MS) in conjunction with robust bioinformatics tools are an excellent way to learn more about protein changes and modifications during disease progression MM. This article addresses in depth the proteomic changes associated with MM disease transformation.