In order to optimize the ~(18)O labeling method, two key aspects, peptide dispersion and trypsin deac tivation were discussed o The addition of Rapigest SF in H_2~('8)O and microwave heating enhanced labeling efficiency of α-casein digested peptides(~(18)O/~(16)O) ratio >99%).Chemical modification with tris(2-carboxyeth yl) phosphine (TCEP) and iodoacetamide (IAA) resulted in trypsin deactivated completely.No significant back-exchange from ~(18)O to ~(16)O was observed after labeling in 6 days.The experiment result with peptide mixture from showed that the improved method could be effectively used to label protein and peptide.

Due to the complexity of proteome samples, comprehensive analysis to characterize all proteins was still not possible with present methodologies. It has been shown that replicate runs could increase the number of identified) proteins. However, the redundancy of protein identifications was high. High-abundant peptides tended to be analyzed repeatedly in different runs. To reduce the redundancy and improve the efficiency of identification), we studied the MS/MS acquisition method of linear ion trap Fourier transform ion cyclotron resonance)-mass spectrometry(LTQ-FT) and an acquisition strategy based on exclusion of precursor ions was developed). It proved that the strategy could extremely reduce the redundancy of MS/MS acquisition and improve) the efficiency of protein identifications.

Posttranslational modifications of antibody products affect their stability,charge distribution,and drug activity and are thus a critical quality attribute.The comprehensive mapping of antibody modifications and different charge isomers(CIs)is of utmost importance,but is challenging.We intended to quanti-tatively characterize the posttranslational modification status of CIs of antibody drugs and explore the impact of posttranslational modifications on charge heterogeneity.The CIs of antibodies were fraction-ated by strong cation exchange chromatography and verified by capillary isoelectric focusing-whole column imaging detection,followed by stepwise structural characterization at three levels.First,the differences between CIs were explored at the intact protein level using a top-down mass spectrometry approach;this showed differences in glycoforms and deamidation status.Second,at the peptide level,common modifications of oxidation,deamidation,and glycosylation were identified.Peptide mapping showed nonuniform deamidation and glycoform distribution among CIs.In total,10 N-glycoforms were detected by peptide mapping.Finally,an in-depth analysis of glycan variants of CIs was performed through the detection of enriched glycopeptides.Qualitative and quantitative analyses demonstrated the dynamics of 24 N-glycoforms.The results revealed that sialic acid modification is a critical factor ac-counting for charge heterogeneity,which is otherwise missed in peptide mapping and intact molecular weight analyses.This study demonstrated the importance of the comprehensive analyses of antibody CIs and provides a reference method for the quality control of biopharmaceutical analysis.

Objective: To study the effect and regulatory mechanism of secreted proteins from PSC on pancreatic ductal adenocarcinoma cells(PANC-1). Methods: Conditioned medium(CM) from pancreatic stellate cells(PSC) was collected through an indirect co-culture method, and PANC-1 cells were cultured separately with CM for 0, 2, and 24 h. The proliferation phenotype of PANC-1 cells under different stimulation periods was detected using the CCK-8 assay. Proteomic analysis was performed to analyze the changes in protein levels of PANC-1 cells, and the most significant protein changes were validated using Western blot. Results: Compared with the control group, the proliferation rate of PANC-1 cells increased after being stimulated by PSC derived CM; The results of proteomic analysis showed that the protein expression of metabolic pathways in PANC-1 cells increased continuously after being cultured in PSC CM for 0, 2, and 24 h. Western blot analysis confirmed an increasing trend of PIK3C2A in PANC-1 cells, indicating that the CM from PSC might promote the proliferation of PANC-1 cells by upregulating the expression of PIK3C2A. Conclusion The CM of PSC may promote the proliferation of PANC-1 cells by upregulating the expression of PIK3C2A, which improves the understanding of the mechanism of interaction between PSCs and pancreatic cancer cells in the tumor microenvironment.