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In order to understand the coordinated proteome changes associated with differentiation of a cultured cell pluripotency model, protein expression changes induced by treatment of NT2 embryonal carcinoma cells with retinoic acid were monitored by mass spectrometry. The relative levels of over 5000 proteins were mapped across distinct cell fractions. Analysis of the chromatin fraction revealed major abundance changes among chromatin proteins and epigenetic pathways between the pluripotent and differentiated states. Protein complexes associated with epigenetic regulation of gene expression, chromatin remodelling (e.g., SWI/SNF, NuRD) and histone-modifying enzymes (e.g., Polycomb, MLL) were found to be extensively regulated. We therefore investigated histone modifications before and after differentiation, observing changes in the global levels of lysine acetylation and methylation across the four canonical histone protein families, as well as among variant histones. We identified the set of proteins with affinity to peptides housing the histone marks H3K4me3 and H3K27me3, and found increased levels of chromatin-associated histone H3 tail trimming following differentiation that correlated with increased expression levels of cathepsin proteases. We further found that inhibition of cathepsins B and D reduces histone H3 clipping. Overall, the work reveals a global reorganization of the cell proteome congruent with differentiation, highlighting the key role of multiple epigenetic pathways, and demonstrating a direct link between cathepsin B and D activity and histone modification. [ABSTRACT FROM AUTHOR]

In order to understand the coordinated proteome changes associated with differentiation of a cultured cell pluripotency model, protein expression changes induced by treatment of NT2 embryonal carcinoma cells with retinoic acid were monitored by mass spectrometry. The relative levels of over 5000 proteins were mapped across distinct cell fractions. Analysis of the chromatin fraction revealed major abundance changes among chromatin proteins and epigenetic pathways between the pluripotent and differentiated states. Protein complexes associated with epigenetic regulation of gene expression, chromatin remodelling (e.g., SWI/SNF, NuRD) and histone-modifying enzymes (e.g., Polycomb, MLL) were found to be extensively regulated. We therefore investigated histone modifications before and after differentiation, observing changes in the global levels of lysine acetylation and methylation across the four canonical histone protein families, as well as among variant histones. We identified the set of proteins with affinity to peptides housing the histone marks H3K4me3 and H3K27me3, and found increased levels of chromatin-associated histone H3 tail trimming following differentiation that correlated with increased expression levels of cathepsin proteases. We further found that inhibition of cathepsins B and D reduces histone H3 clipping. Overall, the work reveals a global reorganization of the cell proteome congruent with differentiation, highlighting the key role of multiple epigenetic pathways, and demonstrating a direct link between cathepsin B and D activity and histone modification.

Ayodele Alaiya,1 Abeer Alshukairi,2 Zakia Shinwari,1 Mariam AL-Fares,3 Jawaher Alotaibi,4 Waleed AlOmaim,3 Ibtihaj Alsharif,5 Razan Bakheet,6 Layla Alharbi,5 Rabab Allam,1 Ayed Asiri,7 Ziad Memish,8 Khaldoun Alromaih,6 Maha Al-Mozaini5 1Proteomics Unit, Stem Cell and Tissue Re-Engineering Program, King Faisal Specialist Hospital and Research Centre, Riyadh, 11211, Saudi Arabia; 2Department of Medicine, King Faisal Specialist Hospital and Research Center, Jeddah, Kingdom of Saudi Arabia; 3Clinical Chemistry Laboratory, Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia; 4Department of Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Kingdom of Saudi Arabia; 5Immunocompromised Host Research Unit, Department of Infection and Immunity, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia; 6Centre for Genomic Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Kingdom of Saudi Arabia; 7Critical Care Services, Al Imam Abdulrahman Al Faisal Hospital, Riyadh, Kingdom of Saudi Arabia; 8Research and Innovation Center, King Saud Medical City, Ministry of Health, Riyadh, Kingdom of Saudi ArabiaCorrespondence: Ayodele AlaiyaProteomics Unit, Stem Cell and Tissue Re-Engineering Program, King Faisal Specialist Hospital and Research Centre, KFSH&RC, BOX 3354, Riyadh, 11211, Saudi ArabiaTel +966 114424178Email aalaiya@kfshrc.edu.saMaha Al-MozainiImmunocompromised Host Research Unit, Department of Infection and Immunity, KFSH&RC, Riyadh, Saudi ArabiaTel +966 114439632Email mmozaini@kfshrc.edu.saPurpose: This study aimed to understand the pathophysiology of host responses to infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/(COVID-19) and Middle East respiratory syndrome coronavirus (MERS-CoV) and to identify proteins for patient stratification with different grades of illness severity.Patients and Methods: Peripheral blood samples from 43 patients with different grades of COVID-19, 7 MERS-CoV patients admitted to the ICU, and 10 healthy subjects were analyzed using label-free quantitative liquid chromatography–mass spectrometry (LC–MS).Results: We identified 193 and 91 proteins that differed significantly between COVID-19 and MERS-CoV sample groups, respectively, and 49 overlapped between datasets. Only 10 proteins are diagnostic of asymptomatic cases, 12 are prognostic of recovery from severe illness, and 28 are prognostic of a fatal outcome of COVID-19. These proteins are implicated in virus-specific/related signaling networks. Notable among the top canonical pathways are humoral immunity, inflammation, acute-phase response signaling, liver X receptor/retinoid X receptor (LXR/RXR) activation, coagulation, and the complement system. Furthermore, we confirmed positive viral shedding in 11.76% of 51 additional peripheral blood samples, indicating that caution should be taken to avoid the possible risk of transfusion of infected blood products.Conclusion: We identified COVID-19 and MERS-CoV protein panels that have potential as biomarkers and might assist in the prognosis of SARS-CoV-2 infection. The identified markers further our understanding of COVID-19 disease pathophysiology and may have prognostic or therapeutic potential in predicting or managing host cell responses to human COVID-19 and MERS-CoV infections.Keywords: COVID-19, SARS-CoV, MERS-CoV, expression proteomics, biomarkers

Background: Human immunodeficiency virus-1 (HIV-1) exploits human host factors to complete its life cycle. Hence, discovery of HIV-regulated host proteins markers would better our understanding of the virus life-cycle and its contribution to pathogenesis and discovery of objective diagnostic and prognostic molecules. Methods: We conducted holistic total proteomics analysis of three closely related study populations including patients with HIV type-1 (HIV-1) and HIV type-2 (HIV-2) as well as HIV-1 elite controllers (HIV-1-EC). Peripheral blood plasma (PBP) samples were subjected to label-free quantitative liquid-chromatography tandem mass-spectrometry (LC–MS/MS). Results: Over 314 unique PBP protein species were identified of which 100 (approx. 32%) were significantly differentially expressed (≥2 to ∞ - fold-change; p

Human immunodeficiency virus-1 (HIV-1) exploits human host factors to complete its life cycle. Hence, discovery of HIV-regulated host proteins markers would better our understanding of the virus life-cycle and its contribution to pathogenesis and discovery of objective diagnostic and prognostic molecules. We conducted holistic total proteomics analysis of three closely related study populations including patients with HIV type-1 (HIV-1) and HIV type-2 (HIV-2) as well as HIV-1 elite controllers (HIV-1-EC). Peripheral blood plasma (PBP) samples were subjected to label-free quantitative liquid-chromatography tandem mass-spectrometry (LC–MS/MS). Over 314 unique PBP protein species were identified of which 100 (approx. 32%) were significantly differentially expressed (≥2 to ∞ - fold-change; p < 0.05) between the three sample cohorts. Of the 100 proteins, 91 were significantly changed between pairs of HIV-1 versus HIV-1-EC, while 83 of the 100 proteins differed significantly between HIV-2 and HIV-1-EC. Interestingly, 76 proteins (87.5%) overlap between the two data sets indicating that majority of these proteins share similar expression changes between HIV-1 and HIV-2 sample groups. Two of the identified proteins, XRCC5 and PSME1, were implicated in the early phase of the pathway network for HIV life cycle, while others were involved in infectious disease and disease of signal transduction. Among them were MAP2K1, RPL23A, RPS3, CALR, PRDX1, SOD2, LMNB1, PHB, and FGB. Despite the high degree of similarity in protein profiles of HIV-1 and HIV-2, six proteins differed significantly including ETFB, PHB2, S100A9, LMO2, PPP3R1 and Vif, a fragment of virion infectivity factor of HIV-1. Additionally, 15 proteins were uniquely expressed, and one of them (LSP1) is present only in HIV-1-EC but absent in HIV1 and HIV-2 and vice versa for the rest 14 proteins. Altogether, we have identified HIV-specific/related protein expression changes that might potentially be capable of early diagnosis and prognosis of HIV diseases and other related infectious diseases. [ABSTRACT FROM AUTHOR]

Pseudomonas putida KT2440 is an important bioplastic-producing industrial microorganism capable of synthesizing the polymeric carbon-rich storage material, polyhydroxyalkanoate (PHA). PHA is sequestered in discrete PHA granules, or carbonosomes, and accumulates under conditions of stress, for example, low levels of available nitrogen. The pha locus responsible for PHA metabolism encodes both anabolic and catabolic enzymes, a transcription factor, and carbonosome-localized proteins termed phasins. The functions of phasins are incompletely understood but genetic disruption of their function causes PHA-related phenotypes. To improve our understanding of these proteins, we investigated the PHA pathways of P.putida KT2440 using three types of experiments. First, we profiled cells grown in nitrogen-limited and nitrogen-excess media using global expression proteomics, identifying sets of proteins found to coordinately increase or decrease within clustered pathways. Next, we analyzed the protein composition of isolated carbonosomes, identifying two new putative components. We carried out physical interaction screens focused on PHA-related proteins, generating a protein-protein network comprising 434 connected proteins. Finally, we confirmed that the outer membrane protein OprL (the Pal component of the Pal-Tol system) localizes to the carbonosome and shows a PHA-related phenotype and therefore is a novel phasin. The combined datasets represent a valuable overview of the protein components of the PHA system in P.putida highlighting the complex nature of regulatory interactions responsive to nutrient stress., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Parkinson's disease ( PD) is an age-related, neurodegenerative motor disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta and presence of α-synuclein-containing protein aggregates. Mutations in the mitochondrial Ser/Thr kinase PTEN-induced kinase 1 ( PINK1) are associated with an autosomal recessive familial form of early-onset PD. Recent studies have suggested that PINK1 plays important neuroprotective roles against mitochondrial dysfunction by phosphorylating and recruiting Parkin, a cytosolic E3 ubiquitin ligase, to facilitate elimination of damaged mitochondria via autophagy-lysosomal pathways. Loss of PINK1 in cells and animals leads to various mitochondrial impairments and oxidative stress, culminating in dopaminergic neuronal death in humans. Using a 2-D polyacrylamide gel electrophoresis proteomics approach, the differences in expressed brain proteome and phosphoproteome between 6-month-old PINK1-deficient mice and wild-type mice were identified. The observed changes in the brain proteome and phosphoproteome of mice lacking PINK1 suggest that defects in signaling networks, energy metabolism, cellular proteostasis, and neuronal structure and plasticity are involved in the pathogenesis of familial PD. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis is a causative agent of tuberculosis ( TB). The ability of M. tuberculosis to be quiescent in the cell has caused the emergence of latent infection. A comprehensive proteomic analysis of M. tuberculosis H37 Rv over three growth phases, namely mid-log (14-day culture), early stationary (28-day culture), and late stationary (50-day culture), was performed in order to study the change in proteome from the mid-log phase to late-stationary phase. Combination methods of two-dimensional electrophoresis (2- DE) and tandem mass spectrometry were used to generate proteome maps of M. tuberculosis at different growth phases. Ten proteins were detected differentially expressed in the late-stationary phase compared with the other two phases. These proteins were SucD, TrpD, and Rv2161c, which belong to metabolic pathway proteins; FadE5, AccD5, DesA1, and Rv1139c are proteins involved in cell wall or lipid biosynthesis, whereas TB21.7 and Rv3224 are conserved hypothetical proteins with unknown function. A surface antigen protein, DesA1, was not detectable in the late-stationary phase, although present in both log and early-stationary phases. The changes in the expression levels of these proteins were in line with the growth environment changes of the bacteria from mid-log phase to late-stationary phase. The information gathered may be valuable in the intervention against latent TB infection. [ABSTRACT FROM AUTHOR]

Highlights: [•] Two-dimensional gel electrophoresis (2-DE) is widely used in proteomics. [•] 2-DE is gaining in popularity. [•] Numerous analytical and bioinformatics techniques may be coupled to 2-DE. [•] New standards in proteomics are established by 2-DE. [Copyright &y& Elsevier]

Abstract: Among the pathological factors known to be associated with Alzheimer disease (AD), oxidative stress induced by the amyloid-β peptide (Aβ) has been demonstrated to play a key role in human brain and animal models of AD. Recently, we reported elevated levels of oxidative damage in the brain of a transgenic (Tg) AD mouse model with Swedish and Indiana familial AD mutations in human amyloid precursor protein (APP) [PDAPP mice, line J20], as evidenced by increased levels of protein carbonyls, 3-nitrotyrosine, and protein-bound 4-hydroxy-2-nonenal. This oxidative damage was dependent on the methionine 35 residue within the Aβ peptide. Further insight into the molecular pathways affected in this Tg model of AD may be gained with discovery-based proteomics studies; therefore, two-dimensional gel-based expression proteomics was performed to compare differences in brain protein levels of J20 Tg mice with non-transgenic (NTg) littermate controls. Based on our studies, we identified six proteins that had significantly increased levels in J20 Tg relative to NTg mice: calcineurin subunit B type 1, ρ GDP-dissociation inhibitor 1, T-complex protein 1 subunit α A, α-enolase, peptidyl-prolyl cis-trans isomerase (Pin-1), and ATP synthase subunit α mitochondrial. Several of these proteins have previously been implicated in in vitro and in vivo models and subjects with AD. Additionally, using redox proteomics analyses we identified two oxidatively-modified proteins: phosphatidylethanolamine-binding protein 1 and Pin-1 with decreased levels of protein 3-nitrotyrosine in J20 Tg mice relative to NTg. Western blotting and immunoprecipitation analyses were used to validate proteomics results. Overall, these studies provide information about changes in the brain proteome as a result of Aβ deposition and clues with which to further direct studies on elucidating AD pathogenesis. [Copyright &y& Elsevier]

Expression proteomics has become, in recent years, a key genome-wide expression approach in fundamental and applied life sciences. This postgenomic technology aims the quantitative analysis of all the proteins or protein forms (the so-called proteome) of a given organism in a given environmental and genetic context. It is a challenge to provide effective training in this area due to its demanding laboratory procedures and laborious computational data analysis. However, the effective training of undergraduates and postgraduates in this field is highly recommended to prepare them for the challenges of postgenomic research and of medical, industrial and other economical activities. Since 2004, the area of Biological Sciences at the Department of Chemical and Biological Engineering of lnstituto Superior Técnico (1ST) has been teaching Expression Proteomics to undergraduate and postgraduate students in three formats: 1) as modules of curricular units (CU), in particular of Functional Genomics and Bioinformatics (FGB), offered as a mandatory CU to 1ST Biological Engineering or Biotechnology Master courses students, or as an elective CU to other MSc courses with a biological component and to the MSc in Information Systems and Computer Engineering; the topic is also part of the PhD program in Biotechnology; 2) as mentored coaching, in which 1ST students integrate ongoing research programs at the Biological Sciences Research Group of IBB at 1ST; and 3) as intensive thematic courses open to the external community. In this article, educational programs and teaching methodologies and tools that we have been using are outlined, from the wet-lab to the laptop. The current role of quantitative proteomics in biological research, with emphasis on microbial stress response and on biomedical and biotechnological applications, is addressed, as a case-study, anchored on our group research activities. [ABSTRACT FROM AUTHOR]

Abstract: Proteomic analysis of plasma is challenging because of its large dynamic range, which prevents the detection of low abundance proteins. Immunodepletion of high abundance proteins, such as albumin and IgG, has emerged as a favored technology to overcome this problem; however its suitability in quantitative expression proteomics has not yet been adequately addressed. In this study, albumin and IgG immunodepletion was evaluated by ELISAs and the reproducibility of depletion was tested with 2-DGE. Depletion of plasma resulted in removal of 62±1.2% of the total protein, 93±1.4% of the albumin (0.43μg/μL, residual), and 94±1.5% of the IgG (0.21μg/μL, residual). These results were confirmed by immunoblotting. Computerized image analysis of 2-D gels using Progenesis SameSpots software revealed an enhancement in the number of visible spots (675–1325), with 10±6% inter-gel variability in spot density. LC–ESI-MS/MS identification of newly resolved protein spots further validated the procedure. An innovative application of the software employed led to identification of 11 proteins lost non-specifically during depletion. This study demonstrates the effectiveness of immunodepletion of albumin and IgG in quantitative 2-DGE-based differential analysis of plasma proteins. [Copyright &y& Elsevier]

Abstract: Cell proteomes are complex, given they consist of several thousand proteins. Two-dimensional electrophoresis (2DE) is unique not only for its ability to simultaneously separate thousands of proteins but also for detecting post- and co-translational modifications, which cannot be predicted from genome sequences. This review will describe the protocols applied to prepare 2D gels properly, and analyse and summarise the major challenges for successful proteome analysis using 2DE, i.e. the ability to analyse very alkaline, hydrophobic and/or low or high M r proteins with high resolution and the ability to detect minor components. Challenges involving sample preparation and solubilisation prior to the first dimension IEF/IPG step will be studied in depth. Sample preparation is crucial in 2DE studies and greatly influences other stages of the technique. It is the aim of this review not only to describe the challenges and limitations of 2DE but also to suggest the avenues, the evolution, the potential and the future of 2DE in proteomics. [Copyright &y& Elsevier]

The human genome has been sequenced, and investigation of its products has become possible in a sequencebased framework. More than 200000 protein species are expressed in the body from ∼30 000 human genes. The term proteome, coined as a linguistic equivalent to the concept of genome, is used to describe the complete set of proteins that is expressed, and modified following expression, by the entire genome in a cell at any one time. Protein types and amounts expressed in a body vary greatly depending upon whether it is healthy or ill. Therefore, proteomics is attracting an increasing interest in its application to better understanding of disease processes, to development ol' new biomarkers for diagnosis and early detection of disease, and to accelerate drug development. There are numerous opportunities for medicine, although it is quite challenging to meet the needs for high sensitivity and high throughput required for disease-related investigations. [ABSTRACT FROM AUTHOR]

Advances in microcapillary liquid chromatography tandem mass spectrometry (LC–MS/MS), in combination with the completion of genome sequencing projects, have opened the door to systematic large-scale mammalian protein expression profiling. The aim of this emerging field is to gain insight into the systems biology of complex multicellular organisms. A recently developed multistep analytical strategy, called PRISM (proteome investigation strategy for mammals), is a promising technology platform for investigating the complex proteome of mammalian tissues. [Copyright &y& Elsevier]

The technical challenge in proving that a given expressed pseudogene is in fact translated into a functional protein is specificity. To circumvent this challenge, one approach is to use PCR in order to generate a series of clones that allow one to exogenously express the pseudogenic protein of interest, either native or fused to a tag, which can facilitate purification, detection, and complementation in both bacterial and mammalian cells. This approach allows an assessment of whether a putative pseudogenic protein possesses enzymatic activity, to identify its subcellular localization and to test its capacity to complement the parental homolog. An alternative approach is to detect the endogenous protein using targeted proteomics analysis and to assess the full range of endogenous RNA isoforms, in order to consider additional coding and noncoding RNA functionality.

Proteomics is the study of proteins, the workhorses of cells. Proteins can be subjected to various post-translational modifications, making them dynamic to external perturbation. Proteomics can be divided into four areas: sequence, structural, functional and interaction and expression proteomics. These different areas used different instrumentations and have different focuses. For example, sequence and structural proteomics mainly focus on elucidating a particular protein sequence and structure, respectively. Meanwhile, functional and interaction proteomics concentrate on protein function and interaction partners, whereas expression proteomics allows the cataloguing of total proteins in any given samples, hence providing a holistic overview of various proteins in a cell. The application of expression proteomics in cancer and crop research is detailed in this chapter. The general workflow of expression proteomics consisting the use of mass spectrometry instrumentation has also been described, and some examples of proteomics studies are also presented.

Diagnostic classification accuracy is critical in expression proteomics to ensure that as many true differences as possible are identified with acceptable false-positive rates. We present a comparison of the diagnostic accuracy of iTRAQ with three label-free methods, peak area, spectral counting, and emPAI, for relative quantification using a spiked proteome standard. We provide the first validation of emPAI for intersample relative quantification and find clear differences among the four quantification approaches that could be considered when designing an experiment. Spectral counting was observed to perform surprisingly well in all regards. Peak area performed best for smaller fold differences and was shown to be capable of discerning a 1.1-fold difference with acceptable specificity and sensitivity. The performance of iTRAQ was dramatically worse than the label-free methods with low abundance proteins. Using the iTRAQ data set for validation, we also demonstrate a novel iTRAQ analysis regime that avoids the use of ratios in significance testing and outperforms a common commercial alternative.