RESUMEN
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.
RESUMEN
We here systematically summarize our practice and its effectiveness in the creation and development of the discipline proteomics in Academy of Military Medical Sciences,and the establishment of a system for identifying and training talented researchers in proteomics.The methods we used to train the researchers and to develop a vigorous team are discussed.These practices contributed much to the development of proteomics in China.
RESUMEN
A derivatization method for the determination of oligosaccharides with high sensitivity by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry(MALDI-TOF-MS) based on a labeling regent 1-(4-Cyanophenyl)-4-piperidinecarbohydrazide(CPH) was proposed. The hydrazones were generated by the reaction of the reducing ends of oligosaccharides and the hydrazide of CPH. The derivatives were directly analyzed by MALDI-TOF-MS. Reaction conditions such as reaction temperature, concentration of acetic acid and excess ratio of CPH regent were optimized. When the oligosaccharides reacted with 100 time excess of CPH at 100 ℃ and in 0.125% acetic acid (V/V), the yield was highest. The results showed the derivatization with CPH increased the sensitivity of MS detection and this method was suitable for the analysis of oligosaccharides′ compositions.
RESUMEN
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.
RESUMEN
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.
RESUMEN
Biological mass spectrometry has been developed for the largE-scale protein identification. The successful identification of protein in proteomic study is based on an effective match of MS data to the sequence in database. Because of the diversity and heterogeneity of protein modification, the experimental data obtained by mass spectrometry does not match the theoretical value sometimes, which makes about 90 percent or more of the tandem mass spectra not be effectively identified. This has become one of the most important technique issues to be resolved in current proteome research. The N-terminal cyclization of peptides, as one of a variety of modification introduced in sample preparation, has been preliminarily studied in this work. The result showed that N-terminal cyclization occurred at the most of the glutamine(Q) or carbamoylmethyl-cysteine(CAM_C) residues and the reaction is often incomplete or partial, both types of peptides could often exist in its respective state at the same time, and the behavior of modified peptides in revered phase chromatography is also changed. The success rate of protein identification could be obviously improved if adding the N-terminal cyclization modification in the database searching. These results will be very helpful in the mass spectrometric data analysis of proteomic study.
RESUMEN
With the progress of technology in separation and identification, proteomics is now widely applied in biomedicine.Because of the powerful ability to research differential protein expression and posttranslational modifications between normal and pathological samples on large scale level, proteomics provides the possibilities to screen biomarkers for diagnosis, therapy or prognosis of diseases.
RESUMEN
Capillary zone electrophoresis (CZE) was used to monitor the whole unfolding process of bovine insulin resulted from the dithiothreitol (DTT) reduction. An off-line matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) was employed to determine the molecular weight of the reaction products at the same time to confirm the observation results obtained by CZE. The structural change during the process of bovine insulin unfolding could be observed directly from the electropherogram and information of protein unfolding could be obtained simultaneously. The results indicated that as an effective tool of monitoring the conformational change of protein,the method of CZE was simple,quick,sensitive and lower sample consumption.
RESUMEN
The mosquito vector of filariasis malayi and its vectorial capacity was investigated In 5 endemic villages in Leshan Prefecture, Sichuan Province. The results showed that the man-biting rate, numan blood index and vectorial capacity of An. lesteri anthropophagus were 0.7, 5.1 and 10.63 times higher than those of An. sinensis. Besides, the natural infection by microfilaria in An, lesteri anthropophagus was also higher than that in An. sinensis by 5 times.From the above result, the authors concluded that An. lesteri anthropophagus was the main vector for transmitting filariasis malayi in the area under study.