Abstract:Analysis of N-glycans released enzymatically from patients' sera or other clinical samples may provide diagnostically and prognostically important information on human disease. Permethylation of these biomolecules simultaneously increases their hydrophobicity and substantially improves their detection parameters in the following mass spectrometric analyses. The overall procedure, from the glycan cleavage to the final mass spectrometric determinations, includes several steps involving extraction, derivatization… Show more
“…Peptides were separated by two-dimensional liquid chromatography (LC). In the first dimension, a manual chromatographic device (97, 98) was used in a modified setting for basic-pH reversed-phase LC fractionation of the complex peptide samples. Briefly, the microcolumn (0.25 by 30 mm) was packed with 2.6-µm Kinetex EVO C 18 core shell particles (Phenomenex) in a fluorinated ethylene propylene tubing (VICI Jour).…”
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 HPVE6andE7oncogenes on host cells, ultimately leading to malignant transformation. Here, we used newly established immortalized human keratinocytes with a well-defined HPV16E6E7expression 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.IMPORTANCEHuman 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 confirmedin vivoin cervical cancer as well as in head and neck cancer patient samples from TCGA data sets.
“…Peptides were separated by two-dimensional liquid chromatography (LC). In the first dimension, a manual chromatographic device (97, 98) was used in a modified setting for basic-pH reversed-phase LC fractionation of the complex peptide samples. Briefly, the microcolumn (0.25 by 30 mm) was packed with 2.6-µm Kinetex EVO C 18 core shell particles (Phenomenex) in a fluorinated ethylene propylene tubing (VICI Jour).…”
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 HPVE6andE7oncogenes on host cells, ultimately leading to malignant transformation. Here, we used newly established immortalized human keratinocytes with a well-defined HPV16E6E7expression 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.IMPORTANCEHuman 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 confirmedin vivoin cervical cancer as well as in head and neck cancer patient samples from TCGA data sets.
“…Both in-gel digestion of proteins and peptide extraction from the gel were performed according to a standard protocol [23]. The extracted peptides were dissolved in 0.1% (v/v) TFA and separated using a manual microgradient device [24]. The device consists of a lab stand as a scaffold and a gas-tight microsyringe connected to a small capillary, prepared from FEP tubing, packed with 3.0 μm core-shell C18-based particles.…”
Background
The availability of tick
in vitro
cell culture systems has facilitated many aspects of tick research, including proteomics. However, certain cell lines have shown a tissue-specific response to infection. Thus, a more thorough characterization of tick cell lines is necessary. Proteomic comparative studies of various tick cell lines will contribute to more efficient application of tick cell lines as model systems for investigation of host-vector-pathogen interactions.
Results
Three cell lines obtained from a hard tick,
Ixodes ricinus
, and two from
I. scapularis
were investigated. A cell mass spectrometry approach (MALDI-TOF MS) was applied, as well as classical proteomic workflows. Using PCA, tick cell line MS profiles were grouped into three clusters comprising IRE/CTVM19 and ISE18, IRE11 and IRE/CTVM20, and ISE6 cell lines. Two other approaches confirmed the results of PCA: in-solution digestion followed by nanoLC-ESI-Q-TOF MS/MS and 2D electrophoresis. The comparison of MS spectra of the cell lines and
I. ricinus
tick organs revealed 29 shared peaks. Of these, five were specific for ovaries, three each for gut and salivary glands, and one for Malpighian tubules. For the first time, characteristic peaks in MS profiles of tick cell lines were assigned to proteins identified in acidic extracts of corresponding cell lines.
Conclusions
Several organ-specific MS signals were revealed in the profiles of tick cell lines.
Electronic supplementary material
The online version of this article (10.1186/s13071-019-3460-5) contains supplementary material, which is available to authorized users.
“…In-gel digestion followed by peptide extraction were carried out according to a protocol by Shevchenko et al [36]. Protein digests were dissolved in 0.1% ( v / v ) trifluoroacetic acid (TFA) and separated using a microgradient device as described in [37]. Briefly, a microcolumn was wetted with 80% acetonitrile (ACN)/0.1% TFA and equilibrated with 0.1% TFA.…”
Simple SummarySturgeon is an ancient and unique fish species. Most of sturgeon are listed as critically endangered species due to habitat alteration and overharvesting. Study of sturgeon reproductive system and sperm is important for aquaculture and conservation programs. Transferrin is recognized as a multiple task protein, positively correlated with spermatogenesis and sperm quality. Thus, we tried to detect transferrin in spermiating and out-of-spawning sterlet reproductive organs and sperm. Two transferrin genes, serotransferrin and melanotransferrin, have been identified in reproductive organs of sterlet males. The serotransferrin was expressed higher in reproductive organs of spermiating than out-of-spawning sterlet males. Furthermore, transferrin was detected in sterlet seminal plasma. This information contributes to the existing information on the variability of transferrin proteins and the potential role of transferrin in chondrostean fishes. AbstractTransferrins are a superfamily of iron-binding proteins and are recognized as multifunctional proteins. In the present study, transcriptomic and proteomic methods were used to identify transferrins in the reproductive organs and sperm of out-of-spawning and spermiating sterlet (Acipenser ruthenus) males. The results showed that seven transferrin transcripts were identified in the transcriptome of sterlet, and these transcripts were qualified as two different transferrin genes, serotransferrin and melanotransferrin, with several isoforms present for serotransferrin. The relative abundance of serotransferrin isoforms was higher in the kidneys and Wolffian ducts in the spermiating males compared to out-of-spawning males. In addition, transferrin was immunodetected in sterlet seminal plasma, but not in sterlet spermatozoa extract. Mass spectrometry identification of transferrin in seminal plasma but not in spermatozoa corroborates immunodetection. The identification of transferrin in the reproductive organs and seminal plasma of sterlet in this study provides the potential function of transferrin during sturgeon male reproduction.
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