Stable isotope labeling by amino acids in cell culture (SILAC) and iodoacetyl tandem mass tag (iodoTMT) are well-implemented mass spectrometry-based approaches for quantification of proteins and for site-mapping of cysteine modification. We describe here a combination of SILAC and iodoTMT to assess ongoing changes in the global proteome and cysteine modification levels using liquid chromatography separation coupled with high-resolution mass spectrometry (LC-MS/MS).

• Klíčová slova
• Cysteine, Global proteome, IodoTMT, Liquid chromatography, Mass spectrometry, Redox, SILAC,
• MeSH
• chromatografie kapalinová metody   MeSH
• cystein metabolismus   MeSH
• izotopové značení metody   MeSH
• oxidace-redukce MeSH
• proteom * metabolismus   MeSH
• proteomika * metody   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystein MeSH
• proteom * MeSH

Affinity purification, combined with mass spectrometry (AP-MS) is considered a pivotal technique in protein-protein interaction studies enabling systematic detection at near physiological conditions. The addition of a quantitative proteomic method, like SILAC metabolic labeling, allows the elimination of non-specifically bound contaminants which greatly increases the confidence of the identified interaction partners. Compared to eukaryotic cells, the SILAC labeling of bacteria has specificities that must be considered. The protocol presented here describes the labeling of bacterial cultures with stable isotope-labeled amino acids, purification of an affinity-tagged protein, and sample preparation for MS measurement. Finally, we discuss the analysis and interpretation of MS data to identify and select the specific partners interacting with the protein of interest. As an example, this workflow is applied to the discovery of potential interaction partners of glyceraldehyde-3-phosphate dehydrogenase in the gram-negative bacterium Francisella tularensis.

• Klíčová slova
• Affinity purification, Bacteria, LC-MS/MS, Protein-protein interactions, SILAC,
• MeSH
• Bacteria metabolismus   MeSH
• chromatografie afinitní MeSH
• hmotnostní spektrometrie metody   MeSH
• izotopové značení metody   MeSH
• proteiny * chemie   MeSH
• proteomika * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny * MeSH

Under normal conditions, the cellular redox status is maintained in a steady state by reduction and oxidation processes. These redox alterations in the cell are mainly sensed by protein thiol residues of cysteines thus regulating protein function. The imbalance in redox homeostasis may therefore regulate protein turnover either directly by redox modulating of transcription factors or indirectly by the degradation of damaged proteins due to oxidation. A new analytical method capable of simultaneously assessing cellular protein expression and cysteine oxidation would provide a valuable tool for the field of cysteine-targeted biology. Here, we show a workflow based on protein quantification using metabolic labeling and determination of cysteine oxidation using reporter ion quantification. We applied this approach to determine protein and redox changes in cells after 5-min, 60-min and 32-h exposure to H2O2, respectively. Based on the functional analysis of our data, we confirmed a biological relevance of this approach and its applicability for parallel mapping of cellular proteomes and redoxomes under diverse conditions. In addition, we revealed a specific pattern of redox changes in peroxiredoxins in a short time-interval cell exposure to H2O2. Overall, our present study offers an innovative, versatile experimental approach to the multifaceted assessment of cellular proteome and its redox status, with broad implications for biomedical research towards a better understanding of organismal physiology and diverse disease conditions.

• Klíčová slova
• Cysteine, Liquid chromatography/mass spectrometry, Peroxiredoxin, Proteome, Redoxome, SILAC-iodoTMT labeling,
• MeSH
• chromatografie kapalinová MeSH
• cystein metabolismus   MeSH
• oxidace-redukce * MeSH
• oxidační stres MeSH
• peroxid vodíku metabolismus   MeSH
• peroxiredoxiny metabolismus   MeSH
• proteom * MeSH
• proteomika * metody   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystein MeSH
• peroxid vodíku MeSH
• peroxiredoxiny MeSH
• proteom * MeSH

Although dendritic cells (DCs) control the priming of the adaptive immunity response, a comprehensive description of their behavior at the protein level is missing. The introduction of the quantitative proteomic technique of metabolic labeling (SILAC) into the field of DC research would therefore be highly beneficial. To achieve this, we applied SILAC labeling to primary bone marow-derived DCs (BMDCs). These cells combine both biological relevance and experimental feasibility, as their in vitro generation permits the use of (13)C/(15)N-labeled amino acids. Interestingly, BMDCs appear to exhibit a very active arginine metabolism. Using standard cultivation conditions, ∼20% of all protein-incorporated proline was a byproduct of heavy arginine degradation. In addition, the dissipation of (15)N from labeled arginine to the whole proteome was observed. The latter decreased the mass accuracy in MS and affected the natural isotopic distribution of peptides. SILAC-connected metabolic issues were shown to be enhanced by GM-CSF, which is used for the differentiation of DC progenitors. Modifications of the cultivation procedure suppressed the arginine-related effects, yielding cells with a proteome labeling efficiency of ≥90%. Importantly, BMDCs generated according to the new cultivation protocol preserved their resemblance to inflammatory DCs in vivo, as evidenced by their response to LPS treatment.

• MeSH
• arginin metabolismus   MeSH
• buňky kostní dřeně metabolismus   MeSH
• dendritické buňky metabolismus   MeSH
• faktor stimulující granulocyto-makrofágové kolonie metabolismus   MeSH
• kultivované buňky MeSH
• molekulární sekvence - údaje MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• prolin metabolismus   MeSH
• proteom * MeSH
• sekvence aminokyselin MeSH
• tandemová hmotnostní spektrometrie MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• arginin MeSH
• faktor stimulující granulocyto-makrofágové kolonie MeSH
• prolin MeSH
• proteom * MeSH

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is well known for its involvement in numerous non-metabolic processes inside mammalian cells. Alternative functions of prokaryotic GAPDH are mainly deduced from its extracellular localization ability to bind to selected host proteins. Data on its participation in intracellular bacterial processes are scarce as there has been to date only one study dealing with this issue. We previously have reported several points of evidence that the GAPDH homolog of Francisella tularensis GapA might also exert additional non-enzymatic functions. Following on from our earlier observations we decided to identify GapA's interacting partners within the bacterial proteome to explore its new roles at intracellular level. The quantitative proteomics approach based on stable isotope labeling of amino acids in cell culture (SILAC) in combination with affinity purification mass spectrometry enabled us to identify 18 proteins potentially interacting with GapA. Six of those interactions were further confirmed by alternative methods. Half of the identified proteins were involved in non-metabolic processes. Further analysis together with quantitative label-free comparative analysis of proteomes isolated from the wild-type strain strain with deleted gapA gene suggests that GapA is implicated in DNA repair processes. Absence of GapA promotes secretion of its most potent interaction partner the hypothetical protein with peptidase propeptide domain (PepSY) thereby indicating that it impacts on subcellular distribution of some proteins.

• Klíčová slova
• Francisella tularensis, SILAC, glyceraldehyde-3-phosphate dehydrogenase, multifunctional enzyme, protein–protein interaction,
• Publikační typ
• časopisecké články MeSH

Cellular senescence is a complex stress response defined as an essentially irreversible cell cycle arrest mediated by the inhibition of cell cycle-specific cyclin dependent kinases. The imbalance in redox homeostasis and oxidative stress have been repeatedly observed as one of the hallmarks of the senescent phenotype. However, a large-scale study investigating protein oxidation and redox signaling in senescent cells in vitro has been lacking. Here we applied a proteome-wide analysis using SILAC-iodoTMT workflow to quantitatively estimate the level of protein sulfhydryl oxidation and proteome level changes in ionizing radiation-induced senescence (IRIS) in hTERT-RPE-1 cells. We observed that senescent cells mobilized the antioxidant system to buffer the increased oxidation stress. Among the antioxidant proteins with increased relative abundance in IRIS, a unique 1-Cys peroxiredoxin family member, peroxiredoxin 6 (PRDX6), was identified as an important contributor to protection against oxidative stress. PRDX6 silencing increased ROS production in senescent cells, decreased their resistance to oxidative stress-induced cell death, and impaired their viability. Subsequent SILAC-iodoTMT and secretome analysis after PRDX6 silencing showed the downregulation of PRDX6 in IRIS affected protein secretory pathways, decreased expression of extracellular matrix proteins, and led to unexpected attenuation of senescence-associated secretory phenotype (SASP). The latter was exemplified by decreased secretion of pro-inflammatory cytokine IL-6 which was also confirmed after treatment with an inhibitor of PRDX6 iPLA2 activity, MJ33. In conclusion, by combining different methodological approaches we discovered a novel role of PRDX6 in senescent cell viability and SASP development. Our results suggest PRDX6 could have a potential as a drug target for senolytic or senomodulatory therapy.

• Klíčová slova
• Cellular senescence, Interleukin 6, Peroxiredoxin 6, Redox proteomics, SILAC-iodoTMT, Senescence-associated secretory phenotype,
• MeSH
• cytokiny * metabolismus   MeSH
• oxidace-redukce MeSH
• oxidační stres MeSH
• peroxiredoxin VI * genetika   metabolismus   MeSH
• stárnutí buněk fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokiny * MeSH
• peroxiredoxin VI * MeSH

The DsbA homolog of Francisella tularensis was previously demonstrated to be required for intracellular replication and animal death. Disruption of the dsbA gene leads to a pleiotropic phenotype that could indirectly affect a number of different cellular pathways. To reveal the broad effects of DsbA, we compared fractions enriched in membrane proteins of the wild-type FSC200 strain with the dsbA deletion strain using a SILAC-based quantitative proteomic analysis. This analysis enabled identification of 63 proteins with significantly altered amounts in the dsbA mutant strain compared to the wild-type strain. These proteins comprise a quite heterogeneous group including hypothetical proteins, proteins associated with membrane structures, and potential secreted proteins. Many of them are known to be associated with F. tularensis virulence. Several proteins were selected for further studies focused on their potential role in tularemia's pathogenesis. Of them, only the gene encoding glyceraldehyde-3-phosphate dehydrogenase, an enzyme of glycolytic pathway, was found to be important for full virulence manifestations both in vivo and in vitro. We next created a viable mutant strain with deleted gapA gene and analyzed its phenotype. The gapA mutant is characterized by reduced virulence in mice, defective replication inside macrophages, and its ability to induce a protective immune response against systemic challenge with parental wild-type strain. We also demonstrate the multiple localization sites of this protein: In addition to within the cytosol, it was found on the cell surface, outside the cells, and in the culture medium. Recombinant GapA was successfully obtained, and it was shown that it binds host extracellular serum proteins like plasminogen, fibrinogen, and fibronectin.

• Klíčová slova
• DsbA, Francisella tularensis, SILAC, glyceraldehyde-3-phosphate dehydrogenase, moonlighting,
• MeSH
• delece genu * MeSH
• faktory virulence analýza   MeSH
• Francisella tularensis enzymologie   imunologie   patogenita   MeSH
• glyceraldehyd-3-fosfátdehydrogenasy nedostatek   metabolismus   MeSH
• krevní proteiny metabolismus   MeSH
• mikrobiální viabilita MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• proteindisulfidisomerasy nedostatek   MeSH
• proteom analýza   MeSH
• salmonelová infekce u zvířat mikrobiologie   patologie   MeSH
• vazba proteinů MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• faktory virulence MeSH
• glyceraldehyd-3-fosfátdehydrogenasy MeSH
• krevní proteiny MeSH
• proteindisulfidisomerasy MeSH
• proteom MeSH

Interferon-induced transmembrane proteins IFITM1 and IFITM3 (IFITM1/3) play a role in both RNA viral restriction and in human cancer progression. Using immunohistochemical staining of FFPE tissue, we identified subgroups of cervical cancer patients where IFITM1/3 protein expression is inversely related to metastasis. Guide RNA-CAS9 methods were used to develop an isogenic IFITM1/IFITM3 double null cervical cancer model in order to define dominant pathways triggered by presence or absence of IFITM1/3 signalling. A pulse SILAC methodology identified IRF1, HLA-B, and ISG15 as the most dominating IFNγ inducible proteins whose synthesis was attenuated in the IFITM1/IFITM3 double-null cells. Conversely, SWATH-IP mass spectrometry of ectopically expressed SBP-tagged IFITM1 identified ISG15 and HLA-B as dominant co-associated proteins. ISG15ylation was attenuated in IFNγ treated IFITM1/IFITM3 double-null cells. Proximity ligation assays indicated that HLA-B can interact with IFITM1/3 proteins in parental SiHa cells. Cell surface expression of HLA-B was attenuated in IFNγ treated IFITM1/IFITM3 double-null cells. SWATH-MS proteomic screens in cells treated with IFITM1-targeted siRNA cells resulted in the attenuation of an interferon regulated protein subpopulation including MHC Class I molecules as well as IFITM3, STAT1, B2M, and ISG15. These data have implications for the function of IFITM1/3 in mediating IFNγ stimulated protein synthesis including ISG15ylation and MHC Class I production in cancer cells. The data together suggest that pro-metastatic growth associated with IFITM1/3 negative cervical cancers relates to attenuated expression of MHC Class I molecules that would support tumor immune escape.

• Klíčová slova
• CAS9 gene editing, Cervical cancer, IFITM1, Interferon, MHC Class I molecule, SILAC mass spectrometry,
• MeSH
• buněčné linie MeSH
• diferenciační antigeny fyziologie   MeSH
• histokompatibilita - antigeny třídy I metabolismus   MeSH
• lidé MeSH
• membránové proteiny fyziologie   MeSH
• nádory děložního čípku metabolismus   MeSH
• proteiny vázající RNA fyziologie   MeSH
• proteosyntéza fyziologie   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• diferenciační antigeny MeSH
• histokompatibilita - antigeny třídy I MeSH
• IFITM3 protein, human MeSH Prohlížeč
• leu-13 antigen MeSH Prohlížeč
• membránové proteiny MeSH
• proteiny vázající RNA MeSH

Although the role of high-risk human papillomaviruses (hrHPVs) as etiological agents in cancer development has been intensively studied during the last decades, there is still the necessity of understanding the impact of the HPV E6 and E7 oncogenes on host cells, ultimately leading to malignant transformation. Here, we used newly established immortalized human keratinocytes with a well-defined HPV16 E6E7 expression cassette to get a more complete and less biased overview of global changes induced by HPV16 by employing transcriptome sequencing (RNA-Seq) and stable isotope labeling by amino acids in cell culture (SILAC). This is the first study combining transcriptome and proteome data to characterize the impact of HPV oncogenes in human keratinocytes in comparison with their virus-negative counterparts. To enhance the informative value and accuracy of the RNA-Seq data, four different bioinformatic workflows were used. We identified potential novel upstream regulators (e.g., CNOT7, SPDEF, MITF, and PAX5) controlling distinct clusters of genes within the HPV-host cell network as well as distinct factors (e.g., CPPED1, LCP1, and TAGLN) with essential functions in cancer. Validated results in this study were compared to data sets from The Cancer Genome Atlas (TCGA), demonstrating that several identified factors were also differentially expressed in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) and HPV-positive head and neck squamous cell carcinomas (HNSCs). This highly integrative approach allows the identification of novel HPV-induced cellular changes that are also reflected in cancer patients, providing a promising omics data set for future studies in both basic and translational research.IMPORTANCE Human papillomavirus (HPV)-associated cancers still remain a big health problem, especially in developing countries, despite the availability of prophylactic vaccines. Although HPV oncogenes have been intensively investigated for decades, a study applying recent advances in RNA-Seq and quantitative proteomic approaches to a precancerous model system with well-defined HPV oncogene expression alongside HPV-negative parental cells has been missing until now. Here, combined omics analyses reveal global changes caused by the viral oncogenes in a less biased way and allow the identification of novel factors and key cellular networks potentially promoting malignant transformation. In addition, this system also provides a basis for mechanistic research on novel key factors regulated by HPV oncogenes, especially those that are confirmed in vivo in cervical cancer as well as in head and neck cancer patient samples from TCGA data sets.

• Klíčová slova
• HPV, RNA-Seq, SILAC, TCGA, cervical cancer, head and neck cancer, integrated analysis,
• MeSH
• adenokarcinom genetika   virologie   MeSH
• dlaždicobuněčné karcinomy hlavy a krku genetika   virologie   MeSH
• genové regulační sítě * MeSH
• karcinogeneze genetika   MeSH
• keratinocyty virologie   MeSH
• lidé MeSH
• lidský papilomavirus 16 genetika   MeSH
• nádorová transformace buněk MeSH
• nádory děložního čípku genetika   virologie   MeSH
• onkogenní proteiny virové genetika   MeSH
• proteom genetika   MeSH
• proteomika MeSH
• spinocelulární karcinom genetika   virologie   MeSH
• stanovení celkové genové exprese MeSH
• transkriptom * MeSH
• výpočetní biologie MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• onkogenní proteiny virové MeSH
• proteom MeSH

Profiling of biological relationships between different molecular layers dissects regulatory mechanisms that ultimately determine cellular function. To thoroughly assess the role of protein post-translational turnover, we devised a strategy combining pulse stable isotope-labeled amino acids in cells (pSILAC), data-independent acquisition mass spectrometry (DIA-MS), and a novel data analysis framework that resolves protein degradation rate on the level of mRNA alternative splicing isoforms and isoform groups. We demonstrated our approach by the genome-wide correlation analysis between mRNA amounts and protein degradation across different strains of HeLa cells that harbor a high grade of gene dosage variation. The dataset revealed that specific biological processes, cellular organelles, spatial compartments of organelles, and individual protein isoforms of the same genes could have distinctive degradation rate. The protein degradation diversity thus dissects the corresponding buffering or concerting protein turnover control across cancer cell lines. The data further indicate that specific mRNA splicing events such as intron retention significantly impact the protein abundance levels. Our findings support the tight association between transcriptome variability and proteostasis and provide a methodological foundation for studying functional protein degradation.

• Klíčová slova
• DIA mass spectrometry, alternative splicing, protein turnover, proteomics, pulsed SILAC,
• MeSH
• alternativní sestřih MeSH
• HeLa buňky MeSH
• hmotnostní spektrometrie MeSH
• izoformy RNA genetika   metabolismus   MeSH
• izotopové značení metody   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• protein - isoformy analýza   metabolismus   MeSH
• proteiny analýza   metabolismus   MeSH
• proteolýza MeSH
• proteomika metody   MeSH
• průběh práce MeSH
• regulace genové exprese u nádorů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• izoformy RNA MeSH
• messenger RNA MeSH
• protein - isoformy MeSH
• proteiny MeSH