Peptide Fragmentation during Nanoelectrospray Ionization

Electrospray ionization (ESI) is considered a soft ionization method, and typically no peptide ion fragmentation is observed. Recently, it has been observed that intensive fragmentation of peptide ions can occur during nanoelectrospray (nanoESI) at special conditions, such as solutions containing high concentrations of salt and relatively low voltage for the spray. In this study, peptide fragmentation during nanoESI has been systematically characterized. The fragmentation phenomenon was observed for a variety of peptides with molecular weights lower than 3000 Da and the types of fragments include a, b, and y ions. For phosphorylated peptides, very little loss of the labile phosphate groups was observed. Solution electrical conductivity (K) and flow rate were identified as the key parameters affecting the degree of peptide fragmentation. Mechanistic studies suggested that very fine first generation charged droplets (∼30 nm in diameter) with high surface electric field (∼1 V/nm) could be formed from nanoESI of highly conductive solutions (K = 0.4 S/m) and at a low flow rate (2 nL/min). It is proposed that solvated peptide ions are ejected with high kinetic energies from the early generations of charged droplets, and the subsequent collisional activation in air induces the peptide fragmentation. The relatively high degree of solvation around the phosphate groups may contribute to the preservation of the phosphorylation during the activation process

Table_1_PSMD2 promotes the progression of bladder cancer and is correlated with immune infiltration.docx

IntroductionPSMD2 plays an oncogenic role in multiple human malignancies, while it is still unclear that the potential roles and underlying mechanisms of PSMD2 in BCa.MethodsThe RNA-seq from TCGA and GTEx database was utilized to preliminarily analyze the expression of PSMD2 in BCa tissues, qRT-PCR was adopted to verify the PSMD2 expression in BCa cell lines. Cox regression analyses were applied to assess the prognostic values of PSMD2 in BCa. GSEA analysis was used to explore the underlying mechanisms of PSMD2. In vitro assays such as wound healing and colony formation assays were applied to determine the carcinogenesis of PSMD2 in BCa. xCell and ssGSEA algorithms were applied to analyze the associations of PSMD2 with TIME.ResultsThe results revealed that in comparison with normal bladder tissues and cell line, PSMD2 was found to be significantly elevated in BCa tissues and cell lines. Elevated expression of PSMD2 can independently predict unfavorable OS for BCa patients. The PSMD2 expression and other clinicopathologic factors were combined to develop a nomogram, which can help to predict OS for BCa patients. GSEA analyses revealed that PSMD2 is correlated with the cell cycle, antigen processing and presentation, JAK-STAT signaling pathway, Toll like receptor signaling pathway, P53 and MAPK signaling pathway. Knockdown of PSMD2 could remarkably inhibit the wound healing and colony formation efficiency of BCa cells. xCell analysis revealed that overexpressed PSMD2 is positively related to the Th2 cells infiltrates and expression levels of immune escape markers, and negatively associated with the infiltrating levels of NK T cell and CD8+ T cell.DiscussionIn conclusion, overexpressed PSMD2 is tightly linked to the immune infiltrates and promotes the progression of BCa.</p

Table_1_Systematical analysis of ferroptosis regulators and identification of GCLM as a tumor promotor and immunological biomarker in bladder cancer.docx

Bladder cancer (BCa) is a life-threaten disease with an increasing incidence with age, and immunotherapy has become an important treatment for BCa, while the efficiency of the immune system declines with age. It is vital to reveal the mechanisms of tumor immune microenvironment (TIME) and identify novel immunotherapy targets for BCa. Through analyzing the RNA-seq of TCGA-BLCA cohort, we distinguished two ferroptosis-related BCa clusters, and we discovered that in comparation with cluster 2, the cluster 1 BCa patients showed higher PD-L1 expression, more unfavorable overall survival and higher tumor stage and grade. XCELL analyses showed that higher level of Th2 cell and Myeloid dendritic cell were enriched in cluster 1, while NK T cell was enriched in cluster 2, and TIDE analysis revealed that cluster 2 was more sensitive to immunotherapy than cluster 1. GSEA analysis implied that Toll-like signaling pathway and JAK_STAT signaling pathway were significantly enriched in cluster 1. Subsequently, through performing bioinformatic analysis and cell experiments, we demonstrated that GCLM is overexpressed in BCa and indicates dismal prognosis, and knockdown of GCLM can significantly suppress the colony formation ability of BCa cells. Furthermore, we also found that GCLM might be correlated with immune infiltration in BCa, and can serve as a tumor promotor and immunological biomarker in BCa, our research showed the vital roles of ferroptosis regulators in TIME of BCa, and GCLM is a latent therapeutic target for cancer immunotherapy.</p

Distribution of OTC species during different pH values.

Distribution of OTC species during different pH values.</p

Adsorption kinetics and isotherms models.

Adsorption kinetics and isotherms models.</p

Effect of temperature on the sorption coefficient (K_d) for OTC sorption on kaolinite.

Effect of temperature on the sorption coefficient (Kd) for OTC sorption on kaolinite.</p

Thermodynamic parameters for OTC adsorption on kaolinite.

Thermodynamic parameters for OTC adsorption on kaolinite.</p

Calculated adsorption coefficients for the OTC species.

Calculated adsorption coefficients for the OTC species.</p

Adsorption isotherms for the OTC adsorption on kaolinite.

Adsorption isotherms for the OTC adsorption on kaolinite.</p

Fourier transform infrared spectroscopic analysis of kaolinite before and after adsorption.

Fourier transform infrared spectroscopic analysis of kaolinite before and after adsorption.</p