33 research outputs found
Integrating transcriptomic and proteomic data for accurate assembly and annotation of genomes
© 2017 Wong et al.; Published by Cold Spring Harbor Laboratory Press. Complementing genome sequence with deep transcriptome and proteome data could enable more accurate assembly and annotation of newly sequenced genomes. Here, we provide a proof-of-concept of an integrated approach for analysis of the genome and proteome of Anopheles stephensi, which is one of the most important vectors of the malaria parasite. To achieve broad coverage of genes, we carried out transcriptome sequencing and deep proteome profiling of multiple anatomically distinct sites. Based on transcriptomic data alone, we identified and corrected 535 events of incomplete genome assembly involving 1196 scaffolds and 868 protein-coding gene models. This proteogenomic approach enabled us to add 365 genes that were missed during genome annotation and identify 917 gene correction events through discovery of 151 novel exons, 297 protein extensions, 231 exon extensions, 192 novel protein start sites, 19 novel translational frames, 28 events of joining of exons, and 76 events of joining of adjacent genes as a single gene. Incorporation of proteomic evidence allowed us to change the designation of more than 87 predicted noncoding RNAs to conventional mRNAs coded by protein-coding genes. Importantly, extension of the newly corrected genome assemblies and gene models to 15 other newly assembled Anopheline genomes led to the discovery of a large number of apparent discrepancies in assembly and annotation of these genomes. Our data provide a framework for how future genome sequencing efforts should incorporate transcriptomic and proteomic analysis in combination with simultaneous manual curation to achieve near complete assembly and accurate annotation of genomes
Non-interferometric methods of phase estimation for application in optical tomography
We describe here two non-interferometric methods for the estimation of the phase of transmitted wavefronts through refracting objects. The phase of the wavefronts obtained is used to reconstruct either the refractive index distribution of the objects or their contours. Refraction corrected reconstructions are obtained by the application of an iterative loop incorporating digital ray tracing for forward propagation and a modified filtered back projection (FBP) for reconstruction. The FBP is modified to take into account non-straight path propagation of light through the object. When the iteration stagnates, the difference between the projection data and an estimate of it obtained by ray tracing through the final reconstruction is reconstructed using a diffraction tomography algorithm. The reconstruction so obtained, viewed as a correction term, is added to the estimate of the object from the loop to obtain an improved final refractive index reconstruction
Phosphotyrosine Profiling Using SILAC
Tyrosine phosphorylation on proteins is an important posttranslational modification that regulates various processes in cells. Mass spectrometry-based phosphotyrosine profiling can reveal tyrosine kinase signaling activity in cells. Using quantitative proteomics strategies such as stable isotope labeling with amino acids in cell culture (SILAC) allows comparison of tyrosine kinase signaling activity across two to –three different conditions. In this book chapter, we discuss the reagents required and a step-by-step protocol to carry out phosphotyrosine profiling using SILAC.</p
Protocol for purification and identification of MHC class I immunopeptidome from cancer cell lines
Major histocompatibility complexes (MHC) play a critical role in immunity by presenting peptides on the cell surface for T cell recognition. Identification of these peptides can be valuable to develop vaccines or immunotherapeutic strategies for infectious diseases and cancers. Mass spectrometry is the only tool available for unbiased identification of the immunopeptidome. Here, we describe a protocol for purification and identification of MHC class I peptides, including in-house purification of anti-MHC-antibody from hybridoma cells and the LC-MS/MS analysis of MHC-I bound peptides. Major histocompatibility complexes (MHC) play a critical role in immunity by presenting peptides on the cell surface for T cell recognition. Identification of these peptides can be valuable to develop vaccines or immunotherapeutic strategies for infectious diseases and cancers. Mass spectrometry is the only tool available for unbiased identification of the immunopeptidome. Here, we describe a protocol for purification and identification of MHC class I peptides, including in-house purification of anti-MHC-antibody from hybridoma cells and the LC-MS/MS analysis of MHC-I bound peptides.</p
Temporal quantitative proteomics reveals proteomic and phosphoproteomic alterations associated with adaptive response to hypoxia in melanoma cells
Hypoxia is a common feature in various solid tumours, including melanoma. Cancer cells in hypoxic environments are resistant to both chemotherapy and radiation. Hypoxia is also associated with immune suppression. Identification of proteins and pathways that regulate cancer cell survival in hypoxic environments can reveal potential vulnerabilities that can be exploited to improve the efficacy of anticancer therapies. We carried out temporal proteomic and phosphoproteomic profiling in melanoma cell lines to identify hypoxia-induced protein expression and phosphorylation changes. By employing a TMT-based quantitative proteomics strategy, we report the identification and quantitation of >7000 proteins and >10,000 phosphosites in melanoma cell lines grown in hypoxia. Proteomics data show metabolic reprogramming as one of the prominent adaptive responses in hypoxia. We identify several novel hypoxia-mediated phosphorylation changes that have not been reported before. They reveal kinase signalling pathways that are potentially involved in modulating cellular response to hypoxia. In addition to known protein expression changes, we identify several novel proteomic alterations associated with adaptive response to hypoxia. We show that cancer cells require the ubiquitin–proteasome system to survive in both normoxia and hypoxia. Inhibition of proteasome activity affects cell survival and may provide a novel therapeutic avenue to target cancer cells in hypoxia. Our study can serve as a valuable resource to pursue novel candidates to target hypoxia in cancers and improve the efficacy of anticancer therapies.</p
A simple organic solvent precipitation method to improve detection of low molecular weight proteins
Mass spectrometry-based proteomics revolutionized global proteomic profiling. Although high molecular weight abundant proteins are readily sampled in global proteomics studies, less abundant low molecular weight proteins are often underrepresented. This includes biologically important classes of low molecular weight proteins including ligands, growth factors, peptide hormones and cytokines. Although extensive fractionation can facilitate achieving better coverage of proteome, it requires additional infrastructure, mass spectrometry time and labour. There is need for a simple method that can selectively deplete high molecular weight abundant proteins and enrich for low molecular weight less abundant proteins to improve their coverage in proteomics studies. We present a simple organic-solvent based protein precipitation method that selectively depletes high molecular weight proteins and enriches low molecular weight proteins in the soluble fraction. Using this strategy, we demonstrate identification of low molecular weight proteins that are generally underrepresented in proteomics datasets. In addition, we show the utility of this approach in identifying functional cleavage products from precursor proteins and low molecular weight short open reading frame proteins encoded by non-coding regions such as lncRNAs and UTRs. As the method does not require additional infrastructure, it can complement existing proteomics workflows to increase detection and coverage of low molecular weight proteins that are less abundant.</p
Protein kinase TgCDPK7 regulates vesicular trafficking and phospholipid synthesis in Toxoplasma gondii.
Apicomplexan parasites are causative agents of major human diseases. Calcium Dependent Protein Kinases (CDPKs) are crucial components for the intracellular development of apicomplexan parasites and are thus considered attractive drug targets. CDPK7 is an atypical member of this family, which initial characterization suggested to be critical for intracellular development of both Apicomplexa Plasmodium falciparum and Toxoplasma gondii. However, the mechanisms via which it regulates parasite replication have remained unknown. We performed quantitative phosphoproteomics of T. gondii lacking TgCDPK7 to identify its parasitic targets. Our analysis lead to the identification of several putative TgCDPK7 substrates implicated in critical processes like phospholipid (PL) synthesis and vesicular trafficking. Strikingly, phosphorylation of TgRab11a via TgCDPK7 was critical for parasite intracellular development and protein trafficking. Lipidomic analysis combined with biochemical and cellular studies confirmed that TgCDPK7 regulates phosphatidylethanolamine (PE) levels in T. gondii. These studies provide novel insights into the regulation of these processes that are critical for parasite development by TgCDPK7
In vivo porcine characterization of atrial lesion safety and efficacy utilizing a circular pulsed-field ablation catheter including assessment of collateral damage to adjacent tissue in supratherapeutic ablation applications.
IntroductionPulsed-field ablation (PFA), an ablative method that causes cell death by irreversible electroporation, has potential safety advantages over radiofrequency ablation and cryoablation. Pulmonary vein (PV) isolation was performed in a porcine model to characterize safety and performance of a novel, fully-integrated biphasic PFA system comprising a multi-channel generator, variable loop circular catheter, and integrated PFA mapping software module.MethodsEight healthy porcine subjects were included. To evaluate safety, multiple ablations were performed, including sites not generally targeted for therapeutic ablation, such as the right inferior PV lumen, right superior PV ostium, and adjacent to the esophagus and phrenic nerve. To evaluate the efficacy, animals were recovered, followed for 30(±3) days, then re-mapped. Gross pathological and histopathological examinations assessed procedural injuries, chronic thrombosis, tissue ablation, penetration depth, healing, and inflammatory response.ResultsAll eight animals survived follow-up. PV narrowing was not observed acutely nor at follow-up, even when ablation was performed deep to the PV ostium. No injury was seen grossly or histologically in adjacent structures. All PVs were durably isolated, confirmed by bidirectional block at re-map procedure. Histological examination showed complete, transmural necrosis around the circumference of the ablated section of right PVs.ConclusionThis preclinical evaluation of a fully-integrated PFA system demonstrated effective and durable ablation of cardiac tissue and PV isolation without collateral damage to adjacent structures, even when ablation was performed in more extreme settings than those used therapeutically. Histological staining confirmed complete transmural cell necrosis around the circumference of the PV ostium at 30 days
Identification and Characterization of Proteins Encoded by Chromosome 12 as Part of Chromosome-centric Human Proteome Project
Chromosome-centric human proteome
project (C-HPP) is a global initiative
to comprehensively characterize proteins encoded by genes across all
human chromosomes by teams focusing on individual chromosomes. Here,
we report mass spectrometry-based identification and characterization
of proteins encoded by genes on chromosome 12. Our study is based
on proteomic profiling of 30 different histologically normal human
tissues and cell types using high-resolution mass spectrometry. In
our analysis, we identified 1,535 proteins encoded by 836 genes on
human chromosome 12. This includes 89 genes that are designated as
“missing proteins” by “neXtProt” as they
did not have any prior evidence either by mass spectrometry or by
antibody-based detection methods. We identified several variant peptides
that reflected coding SNPs annotated in dbSNP database. We also confirmed
the start sites of ∼200 proteins by identifying protein N-terminal
acetylated peptides. We also identified alternative start sites for
11 proteins that were not annotated in public databases until now.
Most importantly, we identified 12 novel protein coding regions on
chromosome 12 using our proteogenomics strategy. All of the 12 regions
have been annotated as pseudogenes in public databases. This study
demonstrates that there is scope for significantly improving annotation
of protein coding genes in the human genome using mass-spectrometry-derived
data. Individual efforts as part of C-HPP initiative should significantly
contribute toward enriching human protein annotation. The data have
been deposited to ProteomeXchange with identifier PXD000561