Mass Spectrometry-based Proteomic Analysis Research Articles

Rare HER2 L796P missense mutation promotes the growth and oncogenic signaling in breast cancer cells.

This research aimed to find potential HER2 mutations that would have an impact on breast cancer and investigate the underlying mechanism. This study first investigated 238 pairs of breast cancer and para-cancerous tissue samples from patients on the targeted next-generation sequencing (tNGS) platform. CCK-8 and clone formation assay were used to investigate whether the mutation exerts proliferative effects on breast cancer cells. In addition, mass spectrometry-based comparative proteomic and phosphoproteomic analyses of the mutation types and wild types of MCF-7 cell lines were carried out. Among the identified mutations, a new mutation HER2 L796P promoted the proliferation of breast cancer cells and had resistance to lapatinib using CCK-8 cell proliferation assay and clone formation assay. The bioinformatic analysis showed that RAS family proteins and ERK phosphorylated proteins significantly increased in the L796P mutant cells. The Gene Ontology (GO) analysis revealed that L796P mutation affected the function of breast cancer at the level of upstream genes in the MAPK and PI3K-AKT-TOR pathways. This study demonstrated that a rare mutation HER2 L796P could be a potential therapeutic target for the clinical management of breast cancer.

Proteomics Analysis Revealed Smad3 as a Potential Target of the Synergistic Antitumor Activity of Disulfiram and Cisplatin in Ovarian Cancer.

Among gynecological cancers, ovarian cancer has a high mortality rate. Cisplatin-based chemotherapy is commonly used for the treatment of ovarian cancer. However, the clinical efficacy of cisplatin in ovarian cancer is limited due to the development of chemo-resistance during treatment. In the study, we aimed to investigate the synergistic anti-cancer activity and targets of the FDA-approved drug disulfiram combined with cisplatin in ovarian cancer. The cell viability was determined by Celltier-Glo luminescent assay. The synergistic anti-cancer activity was assessed by combination index. Cell cycle and apoptosis were detected by flow cytometry. The in vivo anti-tumor activity and side effects were evaluated using a xenografted mice model. The synergistic anti-cancer targets were identified by a mass spectrometry-based proteomics analysis. In this study, we first found that disulfiram synergistically enhanced the anti-tumor activity of cisplatin in chemo-resistant ovarian cancer cells, which was accompanied by the enhanced induction of cellular apoptosis. Secondly, the in vivo study demonstrated that the combination treatment of disulfiram and cisplatin dramatically inhibited tumor growth and had no apparent side effects in ovarian cancer xenografted mice. Finally, proteomics analysis identified SMAD3 as a potential target of disulfiram-cisplatin combined treatment, and the down-regulation of SMAD3 could increase cisplatin-induced cell death in ovarian cancer. Combination treatment of disulfiram and cisplatin synergistically inhibited the growth of ovarian cancer through down-regulating SMAD3. As a repurposed drug, disulfiram could be quickly transformed into a clinic to overcome cisplatin resistance for the treatment of ovarian cancer.

Proteomic Analysis of Pediatric Hemophagocytic Lymphohistiocytosis: a Comparative Study with Healthy Controls, Sepsis, Critical Ill, and Active Epstein-Barr virus Infection to Identify Altered Pathways and Candidate Biomarkers

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome characterized by excessive activation of the immune system, along with uncontrolled proliferation of activated macrophages and lymphocytes. The clinical features of HLH often overlap with the clinical features of other severe inflammatory conditions such as sepsis, hindering accurate and timely diagnosis. In this study, we performed a data-independent acquisition mass spectrometry-based plasma proteomic analysis of 33 pediatric patients with HLH compared with four control groups: 39 healthy children, 43 children with sepsis, 39 children hospitalized in the pediatric intensive care unit without confirmed infections, and 21 children with acute Epstein-Barr virus infection. Proteomic comparisons between the HLH group and each of the control groups showed that HLH was characterized by alterations in complement and coagulation cascades, neutrophil extracellular trap formation, and platelet activation pathways. We identified eight differentially expressed proteins in patients with HLH, including plastin-2 (LCP1), vascular cell adhesion protein 1, fibrinogen beta chain, fibrinogen gamma chain, serum amyloid A-4 protein, extracellular matrix protein 1, apolipoprotein A-I, and albumin. LCP1 emerged as a candidate diagnostic marker for HLH with an area under the curve (AUC) of 0.97 in the original cohort and an AUC of 0.90 (sensitivity = 0.83 and specificity = 1.0) in the validation cohort. Complement C1q subcomponent subunit B was associated with disease severity in patients with HLH. Based on comparisons with multiple control groups, this study provides a proteomic profile and candidate biomarkers of HLH, offering researchers novel information to improve the understanding of this condition.

Proteomics as a Metrological Tool to Evaluate Genome Annotation Accuracy Following De Novo Genome Assembly: A Case Study Using the Atlantic Bottlenose Dolphin (Tursiops truncatus).

The last decade has witnessed dramatic improvements in whole-genome sequencing capabilities coupled to drastically decreased costs, leading to an inundation of high-quality de novo genomes. For this reason, the continued development of genome quality metrics is imperative. Using the 2016 Atlantic bottlenose dolphin NCBI RefSeq annotation and mass spectrometry-based proteomic analysis of six tissues, we confirmed 10,402 proteins from 4711 protein groups, constituting nearly one-third of the possible predicted proteins. Since the identification of larger proteins with more identified peptides implies reduced database fragmentation and improved gene annotation accuracy, we propose the metric NP10, which attempts to capture this quality improvement. The NP10 metric is calculated by first stratifying proteomic results by identifying the top decile (or 10th 10-quantile) of identified proteins based on the number of peptides per protein and then returns the median molecular weight of the resulting proteins. When using the 2016 versus 2012 Tursiops truncatus genome annotation to search this proteomic data set, there was a 21% improvement in NP10. This metric was further demonstrated by using a publicly available proteomic data set to compare human genome annotations from 2004, 2013 and 2016, which showed a 33% improvement in NP10. These results demonstrate that proteomics may be a useful metrological tool to benchmark genome accuracy, though there is a need for reference proteomic datasets across species to facilitate the evaluation of new de novo and existing genome.

Fibrinogen Aα-chain amyloidosis outbreaks in Japanese squirrels (Sciurus lis): a potential disease model.

Fibrinogen Aα-chain amyloidosis is a hereditary systemic amyloidosis characterized by glomerular amyloid depositions, which are derived from the fibrinogen Aα-chain variant in humans. Despite its unique pathology, the pathogenic mechanisms of this disease are only partially understood. This is in part because comparative pathological studies on fibrinogen Aα-chain amyloidosis are currently unavailable as there is a lack of reported cases in animals other than humans. In this study, mass spectrometry-based proteomic analyses of Japanese squirrels (Sciurus lis) that died in five Japanese zoos showed that they developed glomerular-associated fibrinogen Aα-chain amyloidosis with an extremely high incidence rate (29/38 cases, 76.3%). The condition was found to be age-dependent in the Japanese squirrels, with 89% of individuals over 4 years of age affected. Mass spectrometry revealed that the C-terminal region of the fibrinogen Aα-chain was involved in amyloidogenesis in Japanese squirrels as well as humans. No gene variations were identified between amyloid-positive and amyloid-negative squirrels, which contrasted with the available data for humans. The results indicate that fibrinogen Aα-chain amyloidosis is a senile amyloidosis in Japanese squirrels. The results have also provided comparative pathological support that the amyloidogenic C-terminal region of the fibrinogen Aα-chain is involved in the characteristic glomerular pathology, regardless of the animal species. This study elucidates the potential causes of death in Japanese squirrels and will contribute to future comparative pathological studies of fibrinogen Aα-chain amyloidosis. © 2023 The Pathological Society of Great Britain and Ireland.

Lipid peroxidation-derived modification and its effect on the activity of glutathione peroxidase 1

Oxidative stress and the resulting lipid peroxidation are associated with various pathological states, including neurodegenerative diseases and cancer. The end products of lipid peroxidation, such as 4-oxo-2(E)-nonenal (ONE), 4-hydroxy-2(E)-nonenal (HNE), and methylglyoxal (MG), exert several biological effects through modification of various cellular components, including DNA and proteins. Glutathione peroxidase 1 (GPx1) is an intracellular antioxidant enzyme that uses glutathione (GSH) to reduce a variety of peroxides, thereby modulating cellular oxidative stress and redox-mediated responses. GPx1 contains nucleophilic amino acids at its active (one Sec) and GSH-binding (four Arg and one Lys) sites. We found that lipid peroxidation-derived reactive aldehydes (ONE, HNE, and MG) modified the GSH-binding site, resulting in the inhibition of GPx1 activity. Mass spectrometry-based proteomic analysis identified the sites modified by each aldehyde (ONE, 14 sites; HNE, 7 sites; MG, 9 sites). The GSH-binding sites modified were as follows: ONE, Arg57, 103, 184, and 185; HNE, Lys91; MG, Arg103. Upon incubation of GPx1 with each aldehyde, ONE reduced GPx1 activity more significantly than did HNE or MG in a dose- and time-dependent manner. The addition of GSH to GPx1 3 h after incubation with ONE prevented further inhibition by trapping ONE as a ONE-GSH adduct. However, the activity of GPx1 was not restored to the initial level, indicating that ONE modified GPx1 irreversibly. This study suggests that oxidative damage to lipids, resulting in the formation of reactive aldehydes, can amplify cellular oxidative stress via direct inactivation of GPx1, which increases the production of intracellular peroxides.

The boundary of life and death: changes in mitochondrial and cytosolic proteomes associated with programmed cell death of Arabidopsis thaliana suspension culture cells.

Despite the critical role of programmed cell death (PCD) in plant development and defense responses, its regulation is not fully understood. It has been proposed that mitochondria may be important in the control of the early stages of plant PCD, but the details of this regulation are currently unknown. We used Arabidopsis thaliana cell suspension culture, a model system that enables induction and precise monitoring of PCD rates, as well as chemical manipulation of this process to generate a quantitative profile of the alterations in mitochondrial and cytosolic proteomes associated with early stages of plant PCD induced by heat stress. The cells were subjected to PCD-inducing heat levels (10 min, 54°C), with/without the calcium channel inhibitor and PCD blocker LaCl3. The stress treatment was followed by separation of cytosolic and mitochondrial fractions and mass spectrometry-based proteome analysis. Heat stress induced rapid and extensive changes in protein abundance in both fractions, with release of mitochondrial proteins into the cytosol upon PCD induction. In our system, LaCl3 appeared to act downstream of cell death initiation signal, as it did not affect the release of mitochondrial proteins, but instead partially inhibited changes occurring in the cytosolic fraction, including upregulation of proteins with hydrolytic activity. We characterized changes in protein abundance and localization associated with the early stages of heat stress-induced PCD. Collectively, the generated data provide new insights into the regulation of cell death and survival decisions in plant cells.

Dysregulation of the Wnt/β-catenin signaling pathway via Rnf146 upregulation in a VPA-induced mouse model of autism spectrum disorder

Autism spectrum disorder (ASD) is a neurodevelopmental disorder associated with impaired social behavior and communication, repetitive behaviors, and restricted interests. In addition to genetic factors, environmental factors such as prenatal drug exposure contribute to the development of ASD. However, how those prenatal factors induce behavioral deficits in the adult stage is not clear. To elucidate ASD pathogenesis at the molecular level, we performed a high-resolution mass spectrometry-based quantitative proteomic analysis on the prefrontal cortex (PFC) of mice exposed to valproic acid (VPA) in utero, a widely used animal model of ASD. Differentially expressed proteins (DEPs) in VPA-exposed mice showed significant overlap with ASD risk genes, including differentially expressed genes from the postmortem cortex of ASD patients. Functional annotations of the DEPs revealed significant enrichment in the Wnt/β-catenin signaling pathway, which is dysregulated by the upregulation of Rnf146 in VPA-exposed mice. Consistently, overexpressing Rnf146 in the PFC impaired social behaviors and altered the Wnt signaling pathway in adult mice. Furthermore, Rnf146-overexpressing PFC neurons showed increased excitatory synaptic transmission, which may underlie impaired social behavior. These results demonstrate that Rnf146 is critical for social behavior and that dysregulation of Rnf146 underlies social deficits in VPA-exposed mice.

Quantitative proteomics profiling reveals the inhibition of trastuzumab antitumor efficacy by phosphorylated RPS6 in gastric carcinoma.

The anti-HER2 antibody trastuzumab is a standard treatment for gastric carcinoma withHER2 overexpression, but not all patients benefit from treatment with HER2-targeted therapies due to intrinsic and acquired resistance. Thus, more precise predictors for selecting patients to receive trastuzumab therapy are urgently needed. We applied mass spectrometry-based proteomic analysis to 38 HER2-positive gastric tumor biopsies from 19 patients pretreated with trastuzumab (responders n = 10; nonresponders, n = 9) to identify factors that may influence innate sensitivity or resistance to trastuzumab therapy and validated the results in tumor cells and patient samples. Statistical analyses revealed significantly lower phosphorylated ribosomal S6 (p-RPS6) levels in responders than nonresponders, and this downregulation was associated with a durable response and better overall survival after anti-HER2 therapy. High p-RPS6 levels could trigger AKT/mTOR/RPS6 signaling and inhibit trastuzumab antitumor efficacy in nonresponders. We demonstrated that RPS6 phosphorylation inhibitors in combination with trastuzumab effectively suppressed HER2-positive GC cell survival through the inhibition of the AKT/mTOR/RPS6 axis. Our findings provide for the first time a detailed proteomics profile of current protein alterations in patients before anti-HER2 therapy and present a novel and optimal predictor for the response to trastuzumab treatment. HER2-positive GC patients with low expression of p-RPS6 are more likely to benefit from trastuzumab therapy than those with high expression. However, those with high expression of p-RPS6 may benefit from trastuzumab in combination with RPS6 phosphorylation inhibitors.

Long non-coding RNA CASC2 regulates osteoblasts matrix mineralization.

Long non-coding RNAs (lncRNAs) are master regulators of gene expression and have recently emerged as potential innovative therapeutic targets. The deregulation of lncRNA expression patterns has been associated with age-related and noncommunicable diseases in the bone tissue, including osteoporosis and tumors. However, the specific role of lncRNAs in physiological or pathological conditions in the bone tissue still needs to be further clarified, for their exploitation as therapeutic tools. In the present study, we evaluate the potential of the lncRNA CASC2 as a regulator of osteogenic differentiation and mineralization. Results show that CASC2 expression is decreased during osteogenic differentiation of human bone marrow-derived Mesenchymal Stem/Stromal cells (hMSCs). CASC2 knockdown, using small interfering RNA against CASC2 (siCASC2), increases the expression of the late osteogenic marker Bone Sialoprotein (BSP), but does not impact ALP staining level nor the expression of early osteogenic transcripts, including RUNX2 and OPG. Although siCASC2 does not impact hMSC proliferation nor apoptosis, it promotes the mineralization of hMSC cultured under osteogenic-inducing conditions, as shown by the increase of calcium deposits. Mass spectrometry-based proteomic analysis revealed that 89 proteins are regulated by CASC2 at late osteogenic stages, including proteins associated with bone diseases or anthropometric and musculoskeletal traits. Specifically, the Cartilage Oligomeric Matrix Protein (COMP) is highly enhanced by CASC2 knockdown at late stages of osteogenic differentiation, at both transcriptional and protein level. On the other hand, inhibition of COMP impairs osteoblasts mineralization as well as the expression of BSP. The results indicate that lncRNA CASC2 regulates late osteogenic differentiation and mineralization in hMSC via COMP and BSP. In conclusion, this study suggests that targeting lncRNA CASC2 could be a potential approach for modulating bone mineralization.

Comparative transcriptome and proteome analysis explores the antitumor key regulators of ergosterone in H22 tumor-bearing mice

Ergosterone has been proved to have potential antitumor effect on H22 tumor-bearing mice, but the antitumor mechanism and key regulators are still unclear. The current study was aimed to explore the key regulators responsible for antitumor of ergosterone using whole transcriptome and proteome analysis in H22 tumor-bearing mice model. The model of H22 tumor-bearing mice was constructed according to the histopathological data and biochemical parameters. The isolated tumor tissues of different treatment groups were subjected to transcriptomic and proteomic analysis. Our findings demonstrated that 472 differentially expressed genes and 658 proteins were identified in the tumor tissue of different treatment groups through RNA-Seq and liquid chromatography with tandem mass spectrometry-based proteomic analysis, respectively. The combined omics analysis revealed three critical genes/proteins, including Lars2, Sirpα and Hcls1 that could play a role in antitumor pathways. Furthermore, Lars2, Sirpα and Hcls1 genes/proteins, as key regulators of the antitumor effect of ergosterone, were verified by qRT-PCR and western blotting methods, respectively. In summary, our study provides new insights into analysing the antitumor mechanism of ergosterone from the point of view of gene and protein expression and will encourage further development of the antitumor pharmaceutical industry.

A High-Throughput Workflow for FFPE Tissue Proteomics.

Laser capture microdissection (LCM) has become an indispensable tool for mass spectrometry-based proteomic analysis of specific regions obtained from formalin-fixed paraffin-embedded (FFPE) tissue samples in both clinical and research settings. Low protein yields from LCM samples along with laborious sample processing steps present challenges for proteomic analysis without sacrificing protein and peptide recovery. Automation of sample preparation workflows is still under development, especially for samples such as laser-capture microdissected tissues. Here, we present a simplified and rapid workflow using adaptive focused acoustics (AFA) technology for sample processing for high-throughput FFPE-based proteomics. We evaluated three different workflows: standard extraction method followed by overnight trypsin digestion, AFA-assisted extraction and overnight trypsin digestion, and AFA-assisted extraction simultaneously performed with trypsin digestion. The use of AFA-based ultrasonication enables automated sample processing for high-throughput proteomic analysis of LCM-FFPE tissues in 96-well and 384-well formats. Further, accelerated trypsin digestion combined with AFA dramatically reduced the overall processing times. LC-MS/MS analysis revealed a slightly higher number of protein and peptide identifications in AFA accelerated workflows compared to standard and AFA overnight workflows. Further, we did not observe any difference in the proportion of peptides identified with missed cleavages or deamidated peptides across the three different workflows. Overall, our results demonstrate that the workflow described in this study enables rapid and high-throughput sample processing with greatly reduced sample handling, which is amenable to automation.

Biomarkers of Pediatric Cataracts: A Proteomics Analysis of Aqueous Fluid.

Cataracts are among the most common causes of childhood vision loss worldwide. This study seeks to identify differentially expressed proteins in the aqueous humor of pediatric cataract patients. Samples of aqueous humor were collected from pediatric and adult cataract patients and subjected to mass spectrometry-based proteomic analysis. Samples of pediatric cataracts were grouped by subtype and compared to adult samples. Differentially expressed proteins in each subtype were identified. Gene ontology analysis was performed using WikiPaths for each cataract subtype. Seven pediatric patients and ten adult patients were included in the study. Of the pediatric samples, all seven (100%) were male, three (43%) had traumatic cataracts, two (29%) had congenital cataracts, and two (29%) had posterior polar cataracts. Of the adult patients, seven (70%) were female and seven (70%) had predominantly nuclear sclerotic cataracts. A total of 128 proteins were upregulated in the pediatric samples, and 127 proteins were upregulated in the adult samples, with 75 proteins shared by both groups. Gene ontology analysis identified inflammatory and oxidative stress pathways as upregulated in pediatric cataracts. Inflammatory and oxidative stress mechanisms may be involved in pediatric cataract formation and warrant further investigation.

Inhibition of GSK3B phosphorylation improves glucose and lipid metabolism disorder

Hepatic glycolipid metabolism disorder is considered as one of the key pathogenic factors for many chronic diseases. Revealing the molecular mechanism of metabolic disorder and exploring drug targets are crucial for the treatment of glucose and lipid metabolic diseases. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been reported to be associated with the pathogenesis of various metabolic diseases. Herein, GAPDH-knockdown ZFL cells and GAPDH-downregulation zebrafish exhibited significant lipid deposition increase and glycogen reduction, thus inducing glucose and lipid metabolism disorders. Using high-sensitivity mass spectrometry-based proteomic and phosphoproteomic analysis, we identified 6838 proteins and 3738 phosphorylated proteins in GAPDH-knockdown ZFL cells. The protein-protein interaction network and DEPPs analyses showed that gsk3baY216 were involved in lipid and glucose metabolism, which was verified by In vitro study. The enzyme activity analysis and cell staining results showed that HepG2 and NCTC-1469 cells transfected with GSK3BY216F plasmid had significantly lower glucose and insulin levels, the decreased lipid deposition, and the increased glycogen synthesis than those transfected with GSK3BY216E plasmid, suggesting that inhibition of GSK3B phosphorylation could significantly improve GSK3B hyperphosphorylation-induced glucose tolerance impairment and insulin sensitivity reduction. To our knowledge, this is the first multi-omic study of GAPDH-knockdown ZFL cells. This study provides insights into the molecular mechanism of glucose and lipid metabolic disorder, and provides potential targets (kinases) for the treatments of human glucose and lipid metabolic diseases.

Clusters of co-abundant proteins in the brain cortex associated with fronto-temporal lobar degeneration

BackgroundFrontotemporal lobar degeneration (FTLD) is characterized pathologically by neuronal and glial inclusions of hyperphosphorylated tau or by neuronal cytoplasmic inclusions of TDP43. This study aimed at deciphering the molecular mechanisms leading to these distinct pathological subtypes.MethodsTo this end, we performed an unbiased mass spectrometry-based proteomic and systems-level analysis of the middle frontal gyrus cortices of FTLD-tau (n = 6), FTLD-TDP (n = 15), and control patients (n = 5). We validated these results in an independent patient cohort (total n = 24).ResultsThe middle frontal gyrus cortex proteome was most significantly altered in FTLD-tau compared to controls (294 differentially expressed proteins at FDR = 0.05). The proteomic modifications in FTLD-TDP were more heterogeneous (49 differentially expressed proteins at FDR = 0.1). Weighted co-expression network analysis revealed 17 modules of co-regulated proteins, 13 of which were dysregulated in FTLD-tau. These modules included proteins associated with oxidative phosphorylation, scavenger mechanisms, chromatin regulation, and clathrin-mediated transport in both the frontal and temporal cortex of FTLD-tau. The most strongly dysregulated subnetworks identified cyclin-dependent kinase 5 (CDK5) and polypyrimidine tract-binding protein 1 (PTBP1) as key players in the disease process. Dysregulation of 9 of these modules was confirmed in independent validation data sets of FLTD-tau and control temporal and frontal cortex (total n = 24). Dysregulated modules were primarily associated with changes in astrocyte and endothelial cell protein abundance levels, indicating pathological changes in FTD are not limited to neurons.ConclusionsUsing this innovative workflow and zooming in on the most strongly dysregulated proteins of the identified modules, we were able to identify disease-associated mechanisms in FTLD-tau with high potential as biomarkers and/or therapeutic targets.

Colorectal cancer-derived extracellular vesicles containing HSP70 enhance macrophage phagocytosis by up-regulating MARCO expression

In recent years, we have realized that extracellular vesicles (EVs) play a critical role in regulating the intercellular communication between tumor and immune cells in the tumor microenvironment (TME). Tumor-derived extracellular vesicles (TDEVs) profoundly affect the functional changes of tumor-associated macrophages (TAMs) and promote their M2 polarization. Meanwhile, macrophages have a strong phagocytic ability in phagocytosing apoptotic cells. Especially in the course of chemotherapy or radiotherapy, TAMs can phagocytose and remove apoptotic tumor cells, showing anti-inflammatory and pro-tumor effects. However, the underlying mechanisms by which TDEVs regulate macrophage phagocytosis of apoptotic tumor cells have not been fully elucidated. In this study, we focused on the effect of colorectal cancer-derived extracellular vesicles (CRC-EVs) on macrophages. We demonstrated that CRC-EVs enhanced macrophage phagocytosis of apoptotic CRC cells. We then determined that heat shock protein 70 (HSP70) carried in CRC-EVs was responsible for this effect by using mass spectrometry-based proteomic analysis and the CRISPR-Cas9 system. Through transcriptome sequencing of macrophages, we found that the enhanced phagocytosis of macrophages was mainly due to the up-regulation of the macrophage receptor with collagenous structure (MARCO). In addition, we confirmed that the up-regulation of MARCO was mediated by the AKT-STAT3 signaling pathway. Taken together, this study revealed a novel EVs-mediated macrophage phagocytosis mechanism involved in the clearance of apoptotic tumor cells in the TME. Targeting TDEVs may have potential therapeutic applications in tumor treatment.

Chronic Cerebral Hypoperfusion-Induced Disturbed Proteostasis of Mitochondria and MAM Is Reflected in the CSF of Rats by Proteomic Analysis

Declining cerebral blood flow leads to chronic cerebral hypoperfusion which can induce neurodegenerative disorders, such as vascular dementia. The reduced energy supply of the brain impairs mitochondrial functions that could trigger further damaging cellular processes. We carried out stepwise bilateral common carotid occlusions on rats and investigated long-term mitochondrial, mitochondria-associated membrane (MAM), and cerebrospinal fluid (CSF) proteome changes. Samples were studied by gel-based and mass spectrometry-based proteomic analyses. We found 19, 35, and 12 significantly altered proteins in the mitochondria, MAM, and CSF, respectively. Most of the changed proteins were involved in protein turnover and import in all three sample types. We confirmed decreased levels of proteins involved in protein folding and amino acid catabolism, such as P4hb and Hibadh in the mitochondria by western blot. We detected reduced levels of several components of protein synthesis and degradation in the CSF as well as in the subcellular fractions, implying that hypoperfusion-induced altered protein turnover of brain tissue can be detected in the CSF by proteomic analysis.

Toward an Integrated Machine Learning Model of a Proteomics Experiment.

In recent years machine learning has made extensive progress in modeling many aspects of mass spectrometry data. We brought together proteomics data generators, repository managers, and machine learning experts in a workshop with the goals to evaluate and explore machine learning applications for realistic modeling of data from multidimensional mass spectrometry-based proteomics analysis of any sample or organism. Following this sample-to-data roadmap helped identify knowledge gaps and define needs. Being able to generate bespoke and realistic synthetic data has legitimate and important uses in system suitability, method development, and algorithm benchmarking, while also posing critical ethical questions. The interdisciplinary nature of the workshop informed discussions of what is currently possible and future opportunities and challenges. In the following perspective we summarize these discussions in the hope of conveying our excitement about the potential of machine learning in proteomics and to inspire future research.

The Chlamydia trachomatis p-aminobenzoate synthase CADD is a manganese-dependent oxygenase that uses its own amino acid residues as substrates.

CADD (chlamydia protein associating with death domains) is a p-aminobenzoate (pAB) synthase involved in a noncanonical route for tetrahydrofolate biosynthesis in Chlamydia trachomatis. Although previously implicated to employ a diiron cofactor, here, we show that pAB synthesis by CADD requires manganese and the physiological cofactor is most likely a heterodinuclear Mn/Fe cluster. Isotope-labeling experiments revealed that the two oxygen atoms in the carboxylic acid portion of pAB are derived from molecular oxygen. Further, mass spectrometry-based proteomic analyses of CADD-derived peptides demonstrated a glycine substitution at Tyr27, providing strong evidence that this residue is sacrificed for pAB synthesis. Additionally, Lys152 was deaminated and oxidized to aminoadipic acid, supporting its proposed role as a sacrificial amino group donor.

PARP inhibitor olaparib induced differential protein expression in cervical cancer cells

PARP inhibitors are a potential class of chemotherapeutic drugs but PARP inhibitor response has not been explored systematically. We lack a specific understanding of the subset of the proteome preferentially modified in various cancers by PARP inhibitors. Implications of PARP inhibitor and PARP1 in cervical cancer treatment and resistance are not fully elucidated. We conducted a mass spectrometry-based proteomic analysis of cervical cancer Hela cells treated with olaparib. We aimed to identify the alteration in the protein signaling pathway induced by PARP inhibitors beyond the DNA damage response pathway. Our data demonstrate a significant reduction in PARP activity and enhanced cell death after olaparib treatment. We further observed articulated proteomic changes with a significant enrichment of proteins in diverse cellular processes. The differentially expressed proteins were predominantly associated with RNA metabolism, mRNA splicing, processing, and RNA binding. Our data also identified proteins that could probably contribute to survival mechanisms resulting in resistance to PARP inhibitors. Hence, we put forth the overview of proteomic changes induced by PARP inhibitor olaparib in cervical cancer cells. This study highlights the significant proteins modified during PARP inhibition and thus could be a probable target for combination therapies with PARP inhibitors in cervical cancer. Significance•This study provides the overview of proteomic changes induced by PARP inhibitor olaparib in cervical cancer Hela cells.•We demonstrated that Olaparib inhibited PARP1 activity in Hela cells in a dose-dependent manner while no change was observed in the expression of PARP1.•PARP inhibition potentially regulated the RNA metabolism, RNA binding proteins, metastasis-related genes, mitochondrial proteins, transcription factors and regulators, and ubiquitination proteins.•We also identified increased expression of tumor-promoting and drug resistance proteins that could contribute to the resistance mechanism to PARPi therapy.•This approach will be helpful to generate insights into precision oncology for personalized treatment and generate data repositories for future artificial intelligence-based research.