Project description:Quantitative proteome and acetylome analyses were conducted by comparing the acetylation levels of the SM to those of the NM. The collected cellular proteins were extracted, digested, and labeled by different TMTs. For acetylation profiling, the pan acetyl-lysine antibody was employed to enrich the acetylated peptides, which were then submitted into mass spectrometer (MS) for LC–MS/MS analysis. For proteome profiling, the peptide fractions were submitted into MS directly.
Project description:Follicular helper T (Tfh) are a subset of CD4+ T helper cells that provide help to germinal center B cells and mediate the development of long-lived humoral immunity. Tfh cells dysregulation is associated with several major autoimmune diseases. Although recent studies showed Tfh cells differentiation is controlled by the transcription factor Bcl6, cytokines and cell-cell signals, limited information is available on the proteome and post-translational modifications (PTM) of proteins in human Tfh cells. In this study, using TMT labeling technique, antibody-based affinity enrichment and high-resolution LC-MS/MS analysis, we investigated quantitative proteome and acetylome in human naive CD4+ T cells and in vitro induced Tfh (iTfh) cells. In total, we identified 802 up-regulated proteins and 598 down-regulated proteins at the threshold of 1.5 folds in iTfh cells compared to naive CD4+ T cells. With the aid of intensive bioinformatics, biological process, cellular compartment, molecular function, KEGG pathway and protein-protein interaction of these differentially expressed proteins were revealed. Moreover, our acetylome data showed that 22 lysine acetylated proteins are up-regulated and 26 lysine acetylated proteins are down-regulated in iTfh cells compared to the naive CD4+ T cells, among which 11 differentially acetylated lysine residues in core histone were identified, indicating proteins acetylation and epigenetic mechanism are involved in regulating Tfh cells differentiation. These data provide a significant resource for studies of Tfh differentiation and normal and perturbed Tfh cell function.
Project description:Lysine acetylation emerging as a ubiquitous and conserved posttranslational modification plays an important regulatory role in almost every aspect of eukaryotes and prokaryotes. To gain insight into the nature, extent and biological function of lysine acetylation in Beauveria bassiana, a filamentous entomopathogenic fungus, we used immunoaffinity-based acetyl-lysine peptide enrichment integrated with high resolution mass spectrometry to comprehensively characterize lysine acetylated proteins in this fungus. Here we identified a total of 283 proteins with 464 acetylated sites, representing the first acetylproteome reported to date in filamentous fungi. Bioinformatics analysis of this acetylome showed that the acetylated proteins are involved in a wide range of cellular functions, such as metabolism, transcription, and exhibit diverse subcellular localizations. Enrichment of molecular function, biological process, and KEGG pathway implied that identified acetylated proteins of B. bassiana were very important in chromatin organization, ribosome, nucleosome assembly, carbon metabolism, and biosynthesis of secondary metabolites. Moreover, we matched five conserved lysine acetylated motifs containing of KacY, KacH, KacF, FxKac, KacxxxxK and one specific motif KacW in B. bassiana. Taken together, our acetylome analysis revealed a surprising breadth of cellular processes affected by lysine acetylation and also furnishes some fresh intervention nodes for the rational improvement of the friednly entomopathogenic fungus.
Project description:Svinkina T, Gu H, Silva JC, Mertins P, Qiao J, Fereshetian S, Jaffe JD, Kuhn E, Udeshi ND,Carr SA. Mol Cell Proteomics, 2015. Introduction of antibodies specific for acetylated lysine has significantly improved the detection of endogenous acetylation sites by mass spectrometry. Here, we describe a new, commercially available mixture of anti-Kac antibodies and show its utility for in-depth profiling of the acetylome. Specifically, seven complementary monoclones with high specificity for Kac were combined into a final anti-Kac reagent which results in at least a 2-fold increase in identification of Kac peptides over a commonly used Kac antibody. We outline optimal antibody usage conditions, effective offline bRP separation, and use of state-of-the art LC-MS technology for achieving unprecedented coverage of the acetylome. The methods presented in this study enabled the quantification of over 10,000 Kac peptides from over 3000 Kac proteins from a single SILAC labeled sample using 7.5 mg of peptide input per state. These methods result in the deepest coverage of acetylation sites from SAHA treated Jurkat cells. The approach was also applied to breast tumor xenograft samples using isobaric mass tag labeling of peptides (iTRAQ4-plex, TMT6 and TMT10-plex reagents) for quantification. Greater than 6,700 Kac peptides from over 2,300 Kac proteins were quantified using 1 mg of tumor per iTRAQ 4-plex channel.
Project description:By the combination of affinity enrichment and high-resolution LC-MS/MS analysis, large-scale lysine acetylome analysis was performed in picea asperata. Altogether, 1,360 lysine acetylation sites in 717 protein groups were identified. Intensive bioinformatic analysis was then carried out to annotate those lysine acetylated targets, including protein annotation, functional classification, functional enrichment, etc.
Project description:In this study, we detected the first lysine succinylome and acetylome in a vancomycin-intermediate S. aureus (VISA) strain, XN108.Furthermore,Comparative analysis of the protein succinylation and acetylation between XN108-ΔcobB and XN108-WT strain was conducted on the basis of quantitative detection of both protein expression and protein modification
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The objective of this study was to compare transcriptome analysis (RNA-seq) with microarray and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) for HIV-1-infected and uninfected MDMs and to evaluate the optimal protocol for high-throughput data analysis Methods: The mRNA profiles of HIV-1-infected and uninfected MDMs were generated by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 20 million sequence reads per sample to the human genome and identified 60505 transcripts in the HIV-1-infected or uninfected MDMs. RNA-seq data confirmed stable expression of 15 known housekeeping genes, and 10 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8953. Approximately 10% of the transcripts showed differential expression between the HIV-1-infected or uninfected MDMs, with a fold change ≥1.5 and p value <0.05. Altered expression of 48 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Conclusions: Our study represents the detailed analysis of HIV-1-infected macrophages transcriptomes, with biologic replicates, generated by RNA-seq technology. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.