Discover the Post-Translational Modification Proteome Using Mass Spectrometry

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.

ILipo-PseAAC: Identification of Lipoylation Sites Using Statistical Moments and General PseAAC

Lysine Lipoylation is a protective and conserved Post Translational Modification(PTM)in proteomics research like prokaryotes and eukaryotes.It is connected with many biological processes and closely linked with many metabolic diseases.To develop a perfect and accurate classification model for identifying lipoylation sites at the protein level,the computational methods and several other factors play a key role in this purpose.Usually,most of the techniques and different traditional experimental models have a very high cost.They are time-consuming;so,it is required to construct a predictor model to extract lysine lipoylation sites.This study proposes a model that could predict lysine lipoylation sites with the help of a classification method known as Artificial Neural Network(ANN).The ANN algorithm deals with the noise problem and imbalance classification in lipoylation sites dataset samples.As the result shows in ten-fold cross-validation,a brilliant performance is achieved through the predictor model with an accuracy of 99.88%,and also achieved 0.9976 as the highest value of MCC.So,the predictor model is a very useful and helpful tool for lipoylation sites prediction.Some of the residues around lysine lipoylation sites play a vital part in prediction,as demonstrated during feature analysis.The wonderful results reported through the evaluation and prediction of this model can provide an informative and relative explanation for lipoylation and its molecular mechanisms.

Characterization of the P450 Monooxygenase LobP1 as C-32 Hydroxylase in Lobophorin Biosynthesis

Lobophorins(LOBs)belong to a large family of spirotetronate antibiotics with antibacterial and antitumor activities.In this study,we demonstrated the function of LobP1,a P450 monooxygenase encoded in the LOB biosynthetic gene cluster,by in vivo deletion and in vitro biochemical assays.The disruption of lobP1 led to the isolation of three new LOBs derivatives(3-5)and three known ones(6-8)without the hydroxyl group at C-32.LobP1 was shown to have relatively broad substrate scope.Determining the kinetic parameters of LobP1 towards different substrates revealed that LobP1 preferred substrate with a nitrosugar.The new LOBs 3-5 displayed significant antibacterial activities against Bacillus subtilis and Micrococcus luteus with MIC values of 0.125 to 1μg·mL^(-1),and the major product LOB E(6)from the∆lobP1 mutant showed moderate cytotoxic activities against several cancer cell lines.

Bmi-1:At the crossroads of physiological and pathological biology

Bmi-1 is a member of the Polycomb repressor complex 1 that mediates gene silencing by regulating chromatin structure and is indispensable for self-renewal of both normal and cancer stem cells.Despite three decades of research that have elucidated the transcriptional regulation,post-translational modifications and functions of Bmi-1 in regulating the DNA damage response,cellular bioenergetics,and pathologies,the entire potential of a protein with such varied functions remains to be realized.This review attempts to synthesize the current knowledge on Bmi-1 with an emphasis on its role in both normal physiology and cancer.Additionally,since cancer stem cells are emerging as a new paradigm for therapy resistance,the role of Bmi-1 in this perspective is also highlighted.The wide spectrum of malignancies that implicate Bmi-1 as a signature for stemness and oncogenesis also make it a suitable candidate for therapy.Nonetheless,new approaches are vitally needed to further characterize physiological roles of Bmi-1 with the long-term goal of using Bmi-1 as a prognostic marker and a therapeutic target.

Ring-Opening Polymerization of a Benzyl-Protected Cyclic Ester towards Functional Aliphatic Polyester

Monomer design strategy has become a powerful tool to access polymers with desired and diverse functionalities. Here, we designed a novel monomer 2-((benzyloxy)methyl)-1,4-oxathiepan-7-one (BTO) via installing a benzyl ether side chain to the structure of 1,4-oxathiepan-7-one (OTO). The ring-opening polymerization of BTO with Zn1 as the catalyst demonstrated the characteristics of living polymerization with turnover frequency (TOF) up to 2520 h^(−1). With a [BTO]_(0)/[Zn1]_(0)/[I]_(0) feed ratio of 2000/2/1, polymer with high number-average molecular weight (Mn = 536 kDa) and narrow dispersity (Ð = 1.06) was obtained. The produced polymer with a glass transition temperature of -17℃ behaved as an elastomer at room temperature. Consequently, the monomer BTO was copolymerized with _(L)-LA to modulate the mechanical properties of P(L-LA). When the BTO content is 10%, the copolymer exhibits excellent strength (24 MPa) and elongation at break (270%), affording a crystalline, hard, and tough plastic material that combines the high ductility of P(BTO) and the high modulus of P(_(L)-LA). In addition, the oxidation of P(BTO) to P(BTO)-SO_(2) led to an improvement of T_(g) from -17℃ to 38℃. Debenzylation of P(BTO)-SO_(2) afforded P(BTO)-SO_(2)-OH containing free hydroxyl groups. Ultimately, P(BTO) could be hydrolyzed under a base condition to recover the corresponding hydroxyl acid intermediate, which could be used to prepare the monomer again and complete the closed-loop from monomer to polymer to monomer.

Comprehensive Profiling for Histone H4 of Human Liver Cells Using High Resolution LTQ-Orbitrap Mass Spectrometry

The post translational modifications of histone variants are playing an important role in the structure of chromatin, the regulation of gene activities and the diagnosis of diseases, and conducting in-depth researches and discovering new sites depend on new and rational analytical methods to some extent. In this work, the combinatorial method of high resolution LTQ-Orbitrap mass spectrometry and multiple enzymes was employed to identify the post translational modifications （PTMs） of histone H4 of human liver cells. The novel methylation site, argnine 67 （R 67）, was observed besides some sites reported previously such as lysine 31 （K 31）, lysine 44 （K 44）, argnine 55 （R 55） and lysine 59 （K 59） in the global domain. Meanwhile, various combinations of acetylation of lysine 5 （K 5）, lysine 8 （K 8）, lysine 12 （K 12）, lysine 16 （K 16） and methylation of lysine 20 （K 20） in the NH2-terminal tails were also identified after the LC-MS/MS analysis of trypsin, Arg-C, Glu-C and chymotrypsin digests.

Biochemical analysis of granzyme H and elucidation of its structurally important residues involved in substrate and inhibitor binding

Apoptotic cell death plays an important role in the maintenance of the normal physiological state and in the pathogenesis of diseases.Granule exocytosis is the main pathway for the immune elimination of virus-infected cells and tumor cells by cytotoxic T lymphocytes and natural killer cells.In recent study,we have investigated the level of granzyme H in patients with breast cancer and in control subjects using enzymatic method.Our study also included the prediction of different sites of granzyme H that play a role in substrate and inhibitor recognition in apoptosis process by using 3D structural model of the enzyme.The research described the possible post-translational modification sites that may help the enzyme in immune elimination of tumor cells.Our study shows that the level of granzyme H was reduced in patients when compared to normal control subjects.There are a number of amino acids that function as substrate recognition sites in granzyme H.However,inhibitors may inhibit their activity and affect the process of autolysis.

Biochemical reactions in metabolite-protein interaction

Active endogenous metabolites regulate the viability of cells. This process is controlled by a series ofinteractions between small metabolites and large proteins. Previously, several studies had reported thatmetabolite regulates the protein functions, such as diacylglycerol to protein kinase C, lactose regulationof the lac repressor, and HIF-1α stabilization by 2-hydroxyglutarate. However, decades old traditionalbiochemical methods are insufficient to systematically investigate the bio-molecular reactions for a high-throughput discovery. Here, we have reviewed an update on the recently developed chemical proteomicscalled activity-based protein profiling （ABPP）. ABPP is able to identify proteins interacted eithercovalently or non-covalently with metabolites significantly. Thus, ABPP will facilitate the characteriza-tion of specific metabolite regulating; proteins in human disease progression.