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S-Nitrosylation is an important post-translational modification that occurs on cysteine amino acid and regulates signal transduction in diverse cell processes. Dysregulation of protein nitrosylation has shown close association with cardiovascular and neurological diseases, thus demanding further precise and in-depth understanding. Mass spectrometry-based proteomics has been the method of choice for analyzing S-nitrosylated (SNO-) proteins. However, due to their extremely low expression level and rapid turnover rate, quantitative analysis of the S-nitrosylation at the proteomic level remains challenging. Herein, we developed a novel approach termed FluoroTRAQ, which combined the fluorous solid-phase extraction of SNO-peptides and iTRAQ labeling for the quantitative analysis of the SNO-proteome with high sensitivity and specificity. This new analytical strategy was subsequently applied to examine the dynamic SNO-proteome changes of human umbilical vein endothelial cells upon in vitro S-nitrosoglutathione induction. Our data identified a number of novel SNO-proteins and revealed their temporal modulation as validated by biotin switch assay. Our study offered a practical approach for quantitative analysis of protein S-nitrosylation.

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. What is the most favorite and original method developed in your research group? The qualitative and quantitative approaches developed for analysis of PTM proteome. How do you get into this specific field? Could you please share some experiences with our readers? I entered Fudan University in 2003, when the sequencing of human genome was just completed. At that time, the life science entered the post‐genomic era, the era of proteomics. My post‐doctoral work is mainly based on mass spectrometry analysis, which is the core tool for proteome research. Therefore, I began to develop mass spectrometry‐based method for proteome research since then. What is the most important personality for scientific research? Creative, persistence, and collaboration. What's your hobbies? Playing badminton. [ABSTRACT FROM AUTHOR]

Abstract: Hepatitis C virus (HCV) RNA synthesis takes place on a detergent resistant membrane (DRM) structure. To identify potential cellular proteins related to HCV replication complexes (RC), we purified DRMs from HCV subgenomic replicon cells and its parental Huh7 cells. The proteins of DRM fractions were separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Comparing with parental Huh7 cells, 60 proteins were up-regulated while 14 proteins were down-regulated in HCV replicon cells. Ras-GTPase-activating protein binding protein 1 (G3BP1), one of the elevated proteins, was found to be associated with HCV NS5B and knockdown of G3BP1 by siRNA in HCV replicon cells significantly reduced HCV replication, which may indicate it a potential component of HCV RC. These results suggest that HCV viral gene and proteins may regulate the presence of host cellular proteins in DRM, ensure appropriate concentrations of replication components, and hence control the rates or efficiencies of HCV replication. [Copyright &y& Elsevier]

N-myristoylation is one of the most widespread and important lipidation in eukaryotes and some prokaryotes, which is formed by covalently attaching various fatty acids (predominantly myristic acid C14:0) to the N-terminal glycine of proteins. Disorder of N-myristoylation is critically implicated in numerous physiological and pathological processes. Here, we presented a method for purification and comprehensive characterization of endogenous, intact N-glycine lipid-acylated peptides, which combined the n egative s election method for N-terminome and the n ano g raphite f luoride-based s olid- p hase e xtraction method (NeS-nGF SPE). After optimizing experimental conditions, we conducted the first global profiling of the endogenous and heterogeneous modification states for N-terminal glycine, pinpointing the precise sites and their associated lipid moieties. Totally, we obtained 76 N-glycine lipid-acylated peptides, including 51 peptides with myristate (C14:0), 10 with myristoleate (C14:1), 6 with tetradecadienoicate (C14:2), 5 with laurate (C12:0) and 4 with lauroleate (C12:1). Therefore, our proteomic methodology could significantly facilitate precise and in-depth analysis of the endogenous N-myristoylome and its heterogeneity. [Display omitted] • This study presented a strategy, NeS-nGF SPE, to direct analysis of the intact N-myristoylated peptides in mammalian cells. • Both the modification site and its endogenously attached lipid composition was obtained simultaneously in one identification. • We conducted the first global profiling of the endogenous and heterogeneous modification states for N-terminal glycine. [ABSTRACT FROM AUTHOR]

Protein post-translational modifications (PTMs), which include covalent addition of functional groups to proteins and removal of specific amino acid(s) from proteins after synthesis, are often regulated by enzymes and linked to various cellular processes. Identification of enzyme substrates and their modification sites can assist in understanding the control of PTM enzymes on cellular processes, which is important for comprehending physiopathological processes. Due to the rapid advancement of analytical techniques, mass spectrometry (MS)-based proteomic approach has been one of the dominant methods for detecting enzyme-substrate interactions and modification site preferences in biological samples. Here, the development and optimization of MS-based proteomic methodologies in profiling substrates of PTM enzymes are briefly reviewed. [Display omitted] • Current MS-based strategies for substrate profiling of PTM enzymes. • MS-based proteomic methods in site identification of PTM enzymes. • Approaches to introduce enrichment tags for profiling substrates and modification sites of PTM enzymes. • Advances in sample preparation to assist MS-based identification of substrates and modification sites of PTM enzymes. [ABSTRACT FROM AUTHOR]