Mass spectrometry proteomics data are typically evaluated against publicly available annotated sequences, but the proteogenomics approach is a useful alternative. A single genome is commonly utilized in custom proteomic and proteogenomic data analysis. We pose the question of whether utilizing numerous different genome assemblies in a search database would be beneficial. We reanalyzed raw data from the exoprotein fraction of four reference Enterobacterial Repetitive Intergenic Consensus (ERIC) I-IV genotypes of the honey bee bacterial pathogen Paenibacillus larvae and evaluated them against three reference databases (from NCBI-protein, RefSeq, and UniProt) together with an array of protein sequences generated by six-frame direct translation of 15 genome assemblies from GenBank. The wide search yielded 453 protein hits/groups, which UpSet analysis categorized into 50 groups based on the success of protein identification by the 18 database components. Nine hits that were not identified by a unique peptide were not considered for marker selection, which discarded the only protein that was not identified by the reference databases. We propose that the variability in successful identifications between genome assemblies is useful for marker mining. The results suggest that various strains of P. larvae can exhibit specific traits that set them apart from the established genotypes ERIC I-V.
- Klíčová slova
- data mining, diagnostics, mass spectrometry data reanalysis, pathogen diversity, virulence,
- Publikační typ
- časopisecké články MeSH
The ectoparasitic mite Varroa destructor transmits and triggers viral infections that have deleterious effects on honey bee colonies worldwide. We performed a manipulative experiment in which worker bees collected at emergence were exposed to Varroa for 72 h, and their proteomes were compared with those of untreated control bees. Label-free quantitative proteomics identified 77 differentially expressed A. mellifera proteins (DEPs). In addition, viral proteins were identified by orthogonal analysis, and most importantly, Deformed wing virus (DWV) was found at high levels/intensity in Varroa-exposed bees. Pathway enrichment analysis suggested that the main pathways affected included peroxisomal metabolism, cyto-/exoskeleton reorganization, and cuticular proteins. Detailed examination of individual DEPs revealed that additional changes in DEPs were associated with peroxisomal function. In addition, the proteome data support the importance of TGF-β signaling in Varroa-DWV interaction and the involvement of the mTORC1 and Hippo pathways. These results suggest that the effect of DWV on bees associated with Varroa feeding results in aberrant autophagy. In particular, autophagy is selectively modulated by peroxisomes, to which the observed proteome changes strongly corresponded. This study complements previous research with different study designs and suggests the importance of the peroxisome, which plays a key role in viral infections.
- Klíčová slova
- Apis mellifera, DWV, autophagy, host‐pathogen interaction, lipid metabolism,
- MeSH
- hmyzí proteiny metabolismus MeSH
- interakce hostitele a parazita MeSH
- peroxizomy * metabolismus virologie MeSH
- proteom metabolismus analýza MeSH
- proteomika metody MeSH
- RNA-viry * fyziologie MeSH
- signální transdukce MeSH
- Varroidae * virologie MeSH
- včely virologie parazitologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- hmyzí proteiny MeSH
- proteom MeSH
Caspase-9 is the major apical caspase responsible for triggering the intrinsic apoptotic pathway. Our previous study indicated that specific inhibition of caspase-9 caused microscopically evident alterations in appearance of the primary chondrogenic cultures which cannot be explained by decrease in apoptosis. To describe a complex molecular background of this effect, proteomics analysis of control and caspase-9 inhibitor-treated chondrogenic cultures were performed. Proteins were extracted, identified and quantified using LC-MS in both data dependent and data independent acquisition (DIA) mode. While directDIA analysis of diaPASEF data obtained using timsTOF Pro LC-MS system revealed 7849 protein groups (Q-value <0.01), a parallel analysis of iTRAQ-2DLC-MS3 and conventional DIA-MS data identified only 5146 and 4098 protein groups, respectively, showing diaPASEF a superior method for the study. The detailed analysis of diaPASEF data disclosed 236/551 significantly down-/up-regulated protein groups after caspase-9 inhibition, respectively (|log2FC|>0.58, Q value <0.05). Classification of downregulated proteins revealed changes in extracellular matrix organization, collagen metabolism, and muscle system processes. Moreover, deregulations suggest a switch from glycolytic to lipid based metabolism in the inhibited cells. No essential changes were found in the proteins involved in apoptosis. The data indicate new non-apoptotic participation of caspases in chondrocyte homeostasis with potential applications in cartilage pathophysiology.
- Klíčová slova
- caspase-9, chondrogenesis, diaPASEF, micromass cultures, proteomics,
- MeSH
- apoptóza * MeSH
- chondrocyty * metabolismus MeSH
- kaspasa 9 metabolismus farmakologie MeSH
- kaspasy metabolismus farmakologie MeSH
- signální transdukce MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kaspasa 9 MeSH
- kaspasy MeSH
American foulbrood (AFB) is a devastating disease of honey bees. There remains a gap in the understanding of the interactions between the causative agent and host, so we used shotgun proteomics to gain new insights. Nano-LC-MS/MS analysis preceded visual description and Paenibacillus larvae identification in the same individual sample. A further critical part of our methodology was that larvae before capping were used as the model stage. The identification of the virulence factors SplA, PlCBP49, enolase, and DnaK in all P. larvae-positive samples was consistent with previous studies. Furthermore, the results were consistent with the array of virulence factors identified in an in vitro study of P. larvae exoprotein fractions. Although an S-layer protein and a putative bacteriocin were highlighted as important, the microbial collagenase ColA and InhA were not found in our samples. The most important virulence factor identified was isoform of neutral metalloproteinase (UniProt: V9WB82), a major protein marker responsible for the shift in the PCA biplot. This protein is associated with larval decay and together with other virulence factors (bacteriocin) can play a key role in protection against secondary invaders. Overall, this study provides new knowledge on host-pathogen interactions and a new methodical approach to study the disease.
- Klíčová slova
- Apis mellifera, diagnostics with mass spectrometry, host-pathogen interaction, label-free proteomics, neutral metalloproteinase isoforms,
- MeSH
- bakteriociny * metabolismus MeSH
- faktory virulence metabolismus MeSH
- larva MeSH
- Paenibacillus larvae * metabolismus MeSH
- Paenibacillus * metabolismus MeSH
- proteomika MeSH
- tandemová hmotnostní spektrometrie MeSH
- včely MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Spojené státy americké MeSH
- Názvy látek
- bakteriociny * MeSH
- faktory virulence MeSH
Renal cell carcinoma (RCC) represents 2.2% of all cancer incidences; however, prognostic or predictive RCC biomarkers at protein level are largely missing. To support proteomics research of localized and metastatic RCC, we introduce a new library of targeted mass spectrometry assays for accurate protein quantification in malignant and normal kidney tissue. Aliquots of 86 initially localized RCC, 75 metastatic RCC and 17 adjacent non-cancerous fresh frozen tissue lysates were trypsin digested, pooled, and fractionated using hydrophilic chromatography. The fractions were analyzed using LC-MS/MS on QExactive HF-X mass spectrometer in data-dependent acquisition (DDA) mode. A resulting spectral library contains 77,817 peptides representing 7960 protein groups (FDR = 1%). Further, we confirm applicability of this library on four RCC datasets measured in data-independent acquisition (DIA) mode, demonstrating a specific quantification of a substantially increased part of RCC proteome, depending on LC-MS/MS instrumentation. Impact of sample specificity of the library on the results of targeted DIA data extraction was demonstrated by parallel analyses of two datasets by two pan human libraries. The new RCC specific library has potential to contribute to better understanding the RCC development at molecular level, leading to new diagnostic and therapeutic targets.
- Klíčová slova
- assay library, data independent acquisition, mass spectrometry, proteomics, renal cell carcinoma,
- MeSH
- chromatografie kapalinová MeSH
- karcinom z renálních buněk * MeSH
- lidé MeSH
- nádory ledvin * MeSH
- proteom metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- proteom MeSH
Phosphatidylinositol-4-kinases β1 and β2 (PI4Kβ1/PI4Kβ2), which are responsible for phosphorylation of phosphatidylinositol to phosphatidylinositol-4-phosphate, have important roles in plant vesicular trafficking. Moreover, PI4Kβ1/PI4Kβ2 negatively regulates biosynthesis of phytohormone salicylic acid (SA), a key player in plant immune responses. The study focused on the effect of PI4Kβ1/PI4Kβ2 deficiency and SA level on the proteome of microsomal fraction. For that purpose we used four Arabidopsis thaliana genotypes: wild type; double mutant with impaired function of PI4Kβ1/PI4Kβ2 (pi4kβ1/pi4kβ2) exhibiting high SA level; sid2 mutant with impaired SA biosynthesis depending on the isochorismate synthase 1 and triple mutant sid2/pi4kβ1/pi4kβ2. We identified 1797 proteins whose levels were changed between genotypes. We showed that increased SA concentration affected the levels of 473 proteins. This includes typical SA pathway markers but also points to connections between SA pathway and clathrin-independent endocytosis (flotillins) and exocytosis/protein secretion (syntaxins, tetraspanin) to be investigated in future. In contrast to SA, the absence of PI4Kβ1/PI4Kβ2 itself affected only 27 proteins. Among them we identified CERK1, a receptor for chitin. Although PI4Kβ1/PI4Kβ2 deficiency itself did not have a substantial impact on the proteome of the microsomal fraction, our data clearly show that it enhances proteome changes when SA pathway is modulated in parallel.
- Klíčová slova
- Arabidopsis thaliana, label-free proteomics, microsomes, phosphatidylinositol-4-kinase, salicylic acid,
- MeSH
- 1-fosfatidylinositol-4-kinasa genetika metabolismus MeSH
- Arabidopsis * genetika metabolismus MeSH
- fosfatidylinositoly MeSH
- kyselina salicylová MeSH
- proteiny huseníčku * genetika metabolismus MeSH
- proteom MeSH
- regulace genové exprese u rostlin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 1-fosfatidylinositol-4-kinasa MeSH
- fosfatidylinositoly MeSH
- kyselina salicylová MeSH
- proteiny huseníčku * MeSH
- proteom MeSH
In this paper, correlation analysis of protein and mRNA levels in the soil dwelling bacteria Streptomyces coelicolor (S. coelicolor M145) is presented during development of the population as it grew in liquid medium using three biological and two technical replicates, measured during exponential growth, and its entry into the stationary phase. The proteome synthesis time series are compared with the gene expression time series measured previously under identical experimental conditions. Results reveal that about one third of protein/mRNA synthesis profiles are well correlated while another third are correlated negatively. Functional analysis of the highly correlated groups is presented. Based on numerical simulation, the negative correlation between protein and mRNA is shown to be caused by the difference between the rate of translation and protein degradation.
- Klíčová slova
- Streptomyces, computational modeling, mRNA/protein expression, mass spectrometry, proteins functional analysis,
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- messenger RNA genetika metabolismus MeSH
- proteom analýza metabolismus MeSH
- půda chemie MeSH
- regulace genové exprese u bakterií MeSH
- stanovení celkové genové exprese MeSH
- Streptomyces coelicolor genetika růst a vývoj metabolismus MeSH
- transkriptom * MeSH
- vývojová regulace genové exprese * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- messenger RNA MeSH
- proteom MeSH
- půda MeSH
Transgelin is a protein reported to be a marker of several cancers. However, previous studies have shown both up- and down-regulation of transgelin in tumors when compared with non-tumor tissues and the mechanisms whereby transgelin may affect the development of cancer remain largely unknown. Transgelin is especially abundant in smooth muscle cells and is associated with actin stress fibers. These contractile structures participate in cell motility, adhesion, and the maintenance of cell morphology. Here, the role of transgelin in breast cancer is focused on. Initially, the effects of transgelin on cell migration of the breast cancer cell lines, BT 549 and PMC 42, is studied. Interestingly, transgelin silencing increased the migration of PMC 42 cells, but decreased the migration of BT 549 cells. To clarify these contradictory results, the changes in protein abundances after transgelin silencing in these two cell lines are analyzed using quantitative proteomics. The results confirmed the role of transgelin in the migration of BT 549 cells and suggest the involvement of transgelin in apoptosis and small molecule biochemistry in PMC 42 cells. The context-dependent function of transgelin reflects the different molecular backgrounds of these cell lines, which differ in karyotypes, mutation statuses, and proteome profiles.
- Klíčová slova
- apoptosis, breast cancer, cytoskeleton, metastasis, migration, transgelin,
- MeSH
- apoptóza * MeSH
- chromatografie kapalinová MeSH
- down regulace MeSH
- genový knockdown MeSH
- lidé MeSH
- MFC-7 buňky MeSH
- mikrofilamentové proteiny genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- nádory prsu genetika metabolismus patofyziologie MeSH
- pohyb buněk * MeSH
- proteomika MeSH
- regulace genové exprese u nádorů * MeSH
- svalové proteiny genetika metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- umlčování genů MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- mikrofilamentové proteiny MeSH
- svalové proteiny MeSH
- transgelin MeSH Prohlížeč
Biological treatment of many cancers currently targets membrane bound receptors located on a cell surface. To identify novel membrane proteins associated with migration and metastasis of breast cancer cells, a more migrating subpopulation of MDA-MB-231 breast cancer cell line is selected and characterized. A high-resolution quantitative mass spectrometry with SILAC labeling is applied to analyze their surfaceome and it is compared with that of parental MDA-MB-231 cells. Among 824 identified proteins (FDR < 0.01), 128 differentially abundant cell surface proteins with at least one transmembrane domain are found. Of these, i) desmocollin-1 (DSC1) is validated as a protein connected with lymph node status of luminal A breast cancer, tumor grade, and Her-2 status by immunohistochemistry in the set of 96 primary breast tumors, and ii) catechol-O-methyltransferase is successfully verified as a protein associated with lymph node metastasis of triple negative breast cancer as well as with tumor grade by targeted data extraction from the SWATH-MS data of the same set of tissues. The findings indicate importance of both proteins for breast cancer development and metastasis and highlight the potential of biomarker validation strategy via targeted data extraction from SWATH-MS datasets.
- Klíčová slova
- breast cancer, mass spectrometry, metastasis, sequential window acquisition of all theoretical, transmembrane proteins,
- MeSH
- analýza přežití MeSH
- buněčná membrána metabolismus MeSH
- desmokoliny genetika metabolismus MeSH
- fenotyp MeSH
- invazivní růst nádoru MeSH
- katechol-O-methyltransferasa genetika metabolismus MeSH
- lidé MeSH
- lymfatické metastázy patologie MeSH
- nádorové buněčné linie MeSH
- nádory prsu genetika metabolismus patologie MeSH
- pohyb buněk * genetika MeSH
- proteomika * MeSH
- receptor erbB-2 MeSH
- regulace genové exprese u nádorů MeSH
- triple-negativní karcinom prsu genetika metabolismus patologie MeSH
- upregulace genetika MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- COMT protein, human MeSH Prohlížeč
- desmokoliny MeSH
- DSC1 protein, human MeSH Prohlížeč
- ERBB2 protein, human MeSH Prohlížeč
- katechol-O-methyltransferasa MeSH
- receptor erbB-2 MeSH
CRISPR-Cas gene editing holds substantial promise in many biomedical disciplines and basic research. Due to the important functional implications of non-histone chromosomal protein HMG-14 (HMGN1) in regulating chromatin structure and tumor immunity, gene knockout of HMGN1 is performed by CRISPR in cancer cells and the following proteomic regulation events are studied. In particular, DIA mass spectrometry (DIA-MS) is utilized, and more than 6200 proteins (protein- FDR 1%) and more than 82 000 peptide precursors are reproducibly measured in the single MS shots of 2 h. HMGN1 protein deletion is confidently verified by DIA-MS in all of the clone- and dish- replicates following CRISPR. Statistical analysis reveals 147 proteins change their expressions significantly after HMGN1 knockout. Functional annotation and enrichment analysis indicate the deletion of HMGN1 induces histone inactivation, various stress pathways, remodeling of extracellular proteomes, cell proliferation, as well as immune regulation processes such as complement and coagulation cascade and interferon alpha/ gamma response in cancer cells. These results shed new lights on the cellular functions of HMGN1. It is suggested that DIA-MS can be reliably used as a rapid, robust, and cost-effective proteomic-screening tool to assess the outcome of the CRISPR experiments.
- Klíčová slova
- CRISPR-Cas9, HMGN1, clone effect, data independent acquisition, gene deletion, protein function,
- MeSH
- chromatin fyziologie MeSH
- CRISPR-Cas systémy MeSH
- delece genu * MeSH
- editace genu metody MeSH
- HeLa buňky MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- proliferace buněk genetika MeSH
- protein HMGN1 genetika MeSH
- proteomika metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chromatin MeSH
- protein HMGN1 MeSH
