Despite extensive research, the molecular role of AGR2 in the progression and metastasis of colorectal cancer (CRC) has not been fully characterized. We used quantitative mass spectrometry (SWATH MS) to identify differentially expressed proteins in paired CRC cell models of the SW480 and SW620 cell lines in response to AGR2 protein level manipulation. Relying on the results from SWATH MS and subsequent immunochemical validation, we selected NMP3 as the top candidate protein associated with AGR2 in CRC tumour cells in our screen. RT‒qPCR and immunochemical analysis confirmed the involvement of AGR2-mediated regulation of NPM3 at the transcriptional and posttranscriptional levels. Since PD-L1 is a constituent of the NPM3 regulatory axis, we aimed to correlate the changes in PD-L1 to the differential expression of AGR2 in our cell models. We found that AGR2 positively regulates PD-L1 levels in both SW480 and SW620 cell lines; additionally, several different CRC patient transcriptome cohorts confirmed the association of AGR2 with PD-L1. Our work reveals a new AGR2-NPM3 regulatory axis and the involvement of AGR2 in the regulation of PD-L1, which paves the way for the association of AGR2 with immune evasion in CRC cells.
- MeSH
- B7-H1 Antigen * metabolism genetics MeSH
- Nuclear Proteins metabolism genetics MeSH
- Colorectal Neoplasms * genetics metabolism pathology MeSH
- Humans MeSH
- Mucoproteins * metabolism genetics MeSH
- Cell Line, Tumor MeSH
- Nucleophosmin * MeSH
- Oncogene Proteins * metabolism genetics MeSH
- Proteins * metabolism genetics MeSH
- Gene Expression Regulation, Neoplastic * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The unique properties of stem cells to self-renew and differentiate hold great promise in disease modelling and regenerative medicine. However, more information about basic stem cell biology and thorough characterization of available stem cell lines is needed. This is especially essential to ensure safety before any possible clinical use of stem cells or partially committed cell lines. As proteins are the key effector molecules in the cell, the proteomic characterization of cell lines, cell compartments or cell secretome and microenvironment is highly beneficial to answer above mentioned questions. Nowadays, method of choice for large-scale discovery-based proteomic analysis is mass spectrometry (MS) with data-independent acquisition (DIA). DIA is a robust, highly reproducible, high-throughput quantitative MS approach that enables relative quantification of thousands of proteins in one sample. In the current protocol, we describe a specific variant of DIA known as SWATH-MS for characterization of neural stem cell differentiation. The protocol covers the whole process from cell culture, sample preparation for MS analysis, the SWATH-MS data acquisition on TTOF 5600, the complete SWATH-MS data processing and quality control using Skyline software and the basic statistical analysis in R and MSstats package. The protocol for SWATH-MS data acquisition and analysis can be easily adapted to other samples amenable to MS-based proteomics.
- MeSH
- Cell Differentiation MeSH
- Mass Spectrometry methods MeSH
- Humans MeSH
- Neural Stem Cells * chemistry metabolism MeSH
- Proteome analysis MeSH
- Proteomics * methods MeSH
- Quality Control MeSH
- Software * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The neuroprotective E3-ubiquitin ligase CHIP is linked to healthy aging. Here, we present a protocol using a patient-derived iPSC line with a triplication of the α-synuclein gene to produce gene-edited cells isogenic for CHIP. We describe iPSC differentiation into cortical neurons and their identity validation. We then detail mass spectrometry-based approaches (SWATH-MS) to identify dominant changes in the steady state proteome generated by loss of CHIP function. This protocol can be adapted to other proteins that impact proteostasis in neurons. For complete details on the use and execution of this protocol, please refer to Dias et al. (2021).
- MeSH
- Mass Spectrometry MeSH
- Induced Pluripotent Stem Cells * MeSH
- Humans MeSH
- Neurons MeSH
- Proteome genetics MeSH
- Proteomics methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cell therapies represent a promising approach to slow down the progression of currently untreatable neurodegenerative diseases (e.g., Alzheimer's and Parkinson's disease or amyotrophic lateral sclerosis), as well as to support the reconstruction of functional neural circuits after spinal cord injuries. In such therapies, the grafted cells could either functionally integrate into the damaged tissue, partially replacing dead or damaged cells, modulate inflammatory reaction, reduce tissue damage, or support neuronal survival by secretion of cytokines, growth, and trophic factors. Comprehensive characterization of cells and their proliferative potential, differentiation status, and population purity before transplantation is crucial to preventing safety risks, e.g., a tumorous growth due to the proliferation of undifferentiated stem cells. We characterized changes in the proteome and secretome of human neural stem cells (NSCs) during their spontaneous (EGF/FGF2 withdrawal) differentiation and differentiation with trophic support by BDNF/GDNF supplementation. We used LC-MS/MS in SWATH-MS mode for global cellular proteome profiling and quantified almost three thousand cellular proteins. Our analysis identified substantial protein differences in the early stages of NSC differentiation with more than a third of all the proteins regulated (including known neuronal and NSC multipotency markers) and revealed that the BDNF/GDNF support affected more the later stages of the NSC differentiation. Among the pathways identified as activated during both spontaneous and BDNF/GDNF differentiation were the HIF-1 signaling pathway, Wnt signaling pathway, and VEGF signaling pathway. Our follow-up secretome analysis using Luminex multiplex immunoassay revealed significant changes in the secretion of VEGF and IL-6 during NSC differentiation. Our results further demonstrated an increased expression of neuropilin-1 as well as catenin β-1, both known to participate in the regulation of VEGF signaling, and showed that VEGF-A isoform 121 (VEGF121), in particular, induces proliferation and supports survival of differentiating cells.
- Publication type
- Journal Article MeSH
A key component of cardiac ischemia-reperfusion injury (IRI) is the increased generation of reactive oxygen species, leading to enhanced inflammation and tissue dysfunction in patients following intervention for myocardial infarction. In this study, we hypothesized that oxidative stress, due to ischemia-reperfusion, induces senescence which contributes to the pathophysiology of cardiac IRI. We demonstrate that IRI induces cellular senescence in both cardiomyocytes and interstitial cell populations and treatment with the senolytic drug navitoclax after ischemia-reperfusion improves left ventricular function, increases myocardial vascularization, and decreases scar size. SWATH-MS-based proteomics revealed that biological processes associated with fibrosis and inflammation that were increased following ischemia-reperfusion were attenuated upon senescent cell clearance. Furthermore, navitoclax treatment reduced the expression of pro-inflammatory, profibrotic, and anti-angiogenic cytokines, including interferon gamma-induced protein-10, TGF-β3, interleukin-11, interleukin-16, and fractalkine. Our study provides proof-of-concept evidence that cellular senescence contributes to impaired heart function and adverse remodeling following cardiac ischemia-reperfusion. We also establish that post-IRI the SASP plays a considerable role in the inflammatory response. Subsequently, senolytic treatment, at a clinically feasible time-point, attenuates multiple components of this response and improves clinically important parameters. Thus, cellular senescence represents a potential novel therapeutic avenue to improve patient outcomes following cardiac ischemia-reperfusion.
- MeSH
- Humans MeSH
- Reperfusion Injury metabolism MeSH
- Cellular Senescence physiology MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
A specific form of endometrial cancer (EC) can develop in breast cancer patients previously treated with tamoxifen (ET), an antagonist of estrogen receptor (ER) that inhibits proliferation of ER positive breast cancer. ET tumors have a different phenotype than endometrial tumors, which typically develop de novo without previous exposure to tamoxifen (EN). Here we aimed to identify specific protein markers that could serve as specific molecular targets in either phenotype. A set of total 45 formalin-fixed paraffin-embedded (FFPE) endometrial tumor tissues and adjacent myometrium tissue samples were analyzed using LC-MS/MS in SWATH-MS mode. We found that calcyphosin (CAPS) levels were elevated in EN tumors compared to ET tumors. The higher CAPS level in EC tissue invading to myometrium supports its relationship to EC aggressiveness. Further, stathmin (STMN1) levels were found significantly elevated in ET versus EN tumors and significantly associated with patient survival. This finding connects elevated levels of this cell cycle regulating, proliferation-associated protein with tamoxifen exposure. In summary, using SWATH-MS we show that CAPS and STMN1 should be recognized as clinicopathologically different EC markers of which STMN1 is specifically connected with a previous tamoxifen exposition.
- MeSH
- Chromatography, Liquid MeSH
- Humans MeSH
- Endometrial Neoplasms * drug therapy MeSH
- Breast Neoplasms * MeSH
- Stathmin genetics MeSH
- Tamoxifen adverse effects MeSH
- Tandem Mass Spectrometry MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
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.
- MeSH
- Survival Analysis MeSH
- Cell Membrane metabolism MeSH
- Desmocollins genetics metabolism MeSH
- Phenotype MeSH
- Neoplasm Invasiveness MeSH
- Catechol O-Methyltransferase genetics metabolism MeSH
- Humans MeSH
- Lymphatic Metastasis pathology MeSH
- Cell Line, Tumor MeSH
- Breast Neoplasms genetics metabolism pathology MeSH
- Cell Movement * genetics MeSH
- Proteomics * MeSH
- Receptor, ErbB-2 MeSH
- Gene Expression Regulation, Neoplastic MeSH
- Triple Negative Breast Neoplasms genetics metabolism pathology MeSH
- Up-Regulation genetics MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Mass spectrometry raw data repositories, including Metabolomics Workbench and MetaboLights, have contributed to increased transparency in metabolomics studies and the discovery of novel insights in biology by reanalysis with updated computational metabolomics tools. Herein, we reanalyzed the previously published lipidomics data from nine algal species, resulting in the annotation of 1437 lipids achieving a 40% increase in annotation compared to the previous results. Specifically, diacylglyceryl-carboxyhydroxy-methylcholine (DGCC) in Pavlova lutheri and Pleurochrysis carterae, glucuronosyldiacylglycerol (GlcADG) in Euglena gracilis, and P. carterae, phosphatidylmethanol (PMeOH) in E. gracilis, and several oxidized phospholipids (oxidized phosphatidylcholine, OxPC; phosphatidylethanolamine, OxPE; phosphatidylglycerol, OxPG; phosphatidylinositol, OxPI) in Chlorella variabilis were newly characterized with the enriched lipid spectral databases. Moreover, we integrated the data from untargeted and targeted analyses from data independent tandem mass spectrometry (DIA-MS/MS) acquisition, specifically the sequential window acquisition of all theoretical fragment-ion MS/MS (SWATH-MS/MS) spectra, to increase the lipidomic annotation coverage. After the creation of a global library of precursor and diagnostic ions of lipids by the MS-DIAL untargeted analysis, the co-eluted DIA-MS/MS spectra were resolved in MRMPROBS targeted analysis by tracing the specific product ions involved in acyl chain compositions. Our results indicated that the metabolite quantifications based on DIA-MS/MS chromatograms were somewhat inferior to the MS1-centric quantifications, while the annotation coverage outperformed those of the untargeted analysis of the data dependent and DIA-MS/MS data. Consequently, integrated analyses of untargeted and targeted approaches are necessary to extract the maximum amount of metabolome information, and our results showcase the value of data repositories for the discovery of novel insights in lipid biology.
- Publication type
- Journal Article 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.
- MeSH
- Cell Line MeSH
- Antigens, Differentiation physiology MeSH
- Humans MeSH
- Membrane Proteins physiology MeSH
- Histocompatibility Antigens Class I metabolism MeSH
- Uterine Cervical Neoplasms metabolism MeSH
- RNA-Binding Proteins physiology MeSH
- Protein Biosynthesis physiology MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Východiská: Potenciál neoantigénov v indukcii protinádorovej imunitnej odpovede je už desaťročia známy, avšak prvé neoantigénové vakcíny boli vyvinuté len nedávno. Rozvoj genomických a proteomických metód umožnil popis somatických mutácií v nádore a imunogénnosť korešpondujúcich neoantigénov môže byť predpovedaná in silico prípadne in vitro. Kombinácia neoantigénovej vakcinácie so správnym imunologickým postupom umožňuje docieliť regresiu nádoru. Štúdie neoantigénových vakcín na modelových organizmoch dokazujú vysokú účinnosť postupu a dokonca prvé klinické štúdie zaznamenávajú úspech. Cieľ: Cieľom je priblížiť význam neoantigénových vakcín v personalizovanej protinádorovej liečbe a objasniť mechanizmus ich prípravy. Článok popisuje typy mutácií s potenciálom expresie imunogénnych neoantigénov nutných pre tvorbu efektívnej vakcíny a približuje aj ostatné procesy nutné k aktivácii T bunkovej protinádorovej odpovede pacienta. Predovšetkým je zameraný na identifikáciu vysokopravdepodobných neoantigénových sekvencií aplikáciou metód sekvenovania novej generácie a hmotnostnej spektrometrie (mass spectrometry - MS), čo je pre tvorbu vakcíny kľúčové. V článku je popísaný princíp proteogenomickej platformy, ktorá vznikla na našom pracovisku za účelom konfidentnej identifikácie mutantných peptidov v biologickom materiáli. Zmieňuje sa aj o možnosti kvantifikácie neoantigénov MS metódami monitorovania vybraných reakcií (selected reaction monitoring - SRM) a SWATH (sequential windowed acquisition of all theoretical fragment ion spectra). Potenciál neoantigénovej vakcinácie v protinádorovej terapii dokazujú úspešné klinické štúdie zhrnuté v závere článku.
Background: Although immune responses to "cancer neoantigens" have been known for decades, the first neoantigen vaccines emerged only very recently. Current developments in genomics and proteomics have enabled descriptions of tumor mutational landscapes, and the immunogenicity of corresponding neoantigens can now be predicted either in silico or in vitro. Cancer regression could be achieved via a combination of neoantigen vaccination and an appropriate immunology approach. Research in model organisms and the results of initial clinical trials of neoantigen vaccines have shown them to be effective. Purpose: We aim to emphasize the importance of neoantigen vaccines in personalized cancer treatment and describe their preparation. We summarize mutations leading to expression of an immunogenic antigen necessary for vaccine development. The processes leading to activation of T-cell anticancer immunity in a patient are briefly introduced. We especially focus on the identification of high confidence neoantigens by next-generation sequencing (NGS) and mass spectrometry (MS), which is key element in the process of designing neoantigen vaccines. Briefly, we describe a proteogenomic platform for confident identification of mutant peptides in biological material. We mention the possibility of neoantigen quantification in biological material using mass spectrometry such as SRM (selected reaction monitoring) and SWATH (sequential windowed acquisition of all theoretical fragment ion spectra). Successful clinical studies demonstrating the potential of neoantigen vaccination in personalized cancer treatment are summarized at the end of the paper.
- MeSH
- Antigens, Neoplasm therapeutic use MeSH
- Humans MeSH
- Neoplasms * immunology therapy MeSH
- Cancer Vaccines * MeSH
- Vaccination methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
