Herein, we developed a fractionation-free negative enriching method incorporating methylamidation, siteselective dimethylation and aldehyde resin coupling(MADMAR) for in-depth C-terminome analysis. The methylamidation...Herein, we developed a fractionation-free negative enriching method incorporating methylamidation, siteselective dimethylation and aldehyde resin coupling(MADMAR) for in-depth C-terminome analysis. The methylamidation blocked the free carboxyl group on proteins first, followed by Lys C digestion of methylamidated proteins. Then, the site-selective dimethylation blocked the N-terminal amino group of the digested peptides without affecting the amino groups of lysine. Finally, the aldehyde resin was used to capture non-C-terminal peptides containing amino groups from lysine, while leaving the C-terminal peptides without free amino group in the supernatant for its analysis. We identified 1359 database-annotated protein C-termini from 50 μg He La proteins, which was 74% more than our previous method based on aldehyde resin. Moreover, 279 protein neo-C-termini were identified.展开更多
Protein post-translational modifications(PTMs)are chemical modifications on proteins.PTMs play a key role in many cellular processes by influencing the structure of proteins and dynamically regulating their functions....Protein post-translational modifications(PTMs)are chemical modifications on proteins.PTMs play a key role in many cellular processes by influencing the structure of proteins and dynamically regulating their functions.Therefore,characterizing PTMs at proteome level is critical to provide invaluable insight into the functions of proteins underlying different biological processes.Advances in modern proteomics technologies including sample preparation,chromatography separation as well as mass spectrometry have propelled the PTMs proteome to further depths.During the past decade,to better examine the PTMs with high sensitivity and selectivity,our group have developed a series of MS-based novel analytical approaches to study the protein PTMs.Herein,we mainly introduce these approaches developed by our group and discuss how to overcome the technical obstacles of studying various protein PTMs with mass spectrometry.展开更多
基金supported by the National Key Research and Development Program of China (No. 2017YFA0505001)National Natural Science Foundation of China (No. 21974025)the project of Shanghai Key Laboratory of Kidney and Blood Purification。
文摘Herein, we developed a fractionation-free negative enriching method incorporating methylamidation, siteselective dimethylation and aldehyde resin coupling(MADMAR) for in-depth C-terminome analysis. The methylamidation blocked the free carboxyl group on proteins first, followed by Lys C digestion of methylamidated proteins. Then, the site-selective dimethylation blocked the N-terminal amino group of the digested peptides without affecting the amino groups of lysine. Finally, the aldehyde resin was used to capture non-C-terminal peptides containing amino groups from lysine, while leaving the C-terminal peptides without free amino group in the supernatant for its analysis. We identified 1359 database-annotated protein C-termini from 50 μg He La proteins, which was 74% more than our previous method based on aldehyde resin. Moreover, 279 protein neo-C-termini were identified.
基金the National Key Research and Development Pro-gram of China(2016YFA0501303)NSF of China(Grants 21974025 and 31670835)Shanghai Key Laboratory of Clinical Geriatric Medicine(13dz2260700)for financial support.
文摘Protein post-translational modifications(PTMs)are chemical modifications on proteins.PTMs play a key role in many cellular processes by influencing the structure of proteins and dynamically regulating their functions.Therefore,characterizing PTMs at proteome level is critical to provide invaluable insight into the functions of proteins underlying different biological processes.Advances in modern proteomics technologies including sample preparation,chromatography separation as well as mass spectrometry have propelled the PTMs proteome to further depths.During the past decade,to better examine the PTMs with high sensitivity and selectivity,our group have developed a series of MS-based novel analytical approaches to study the protein PTMs.Herein,we mainly introduce these approaches developed by our group and discuss how to overcome the technical obstacles of studying various protein PTMs with mass spectrometry.
