Project description:Identification of 187AA unique peptides by mass spectrometry and detection of 187AA by mass spectrometry in 5 human cells (293F 293T SK-hep1 ESC-derived cardiomyocytes) and 1mouse cell(R1).
Project description:Identification of 187AA unique peptides by mass spectrometry and detection of 187AA by mass spectrometry in 4 human cells (293T, SK-hep1, ESC-derived cardiomyocytes) and 1mouse cell(R1).
Project description:Identification of 187AA unique peptides by mass spectrometry and detection of 187AA by mass spectrometry in human cells (GZF2-iPSC, GZF2-hep, 293T, SK-hep1, ESC-derived cardiomyocytes, Hep-G2 and Hela).
Project description:Identification of 187AA unique peptides by mass spectrometry and detection of 187AA by mass spectrometry in human cells (GZF2-iPSC, GZF2-hep, 293T, SK-hep1, ESC-derived cardiomyocytes, Hep-G2 and Hela).
Project description:Identification of 187AA unique peptides by mass spectrometry and detection of 187AA by mass spectrometry in eight human cells (GZF2-iPSC, GZF2-hep, 293T, SK-hep1, ESC-derived cardiomyocytes, Hep-G2, QSG-7701 and Hela).
Project description:The challenge of discovering a completely new human tumor virus of unknown phylogeny or sequence depends on detecting viral molecules and differentiating them from host-derived molecules in the virus-associated neoplasm. We developed differential peptide subtraction (DPS) using differential mass-spectrometry (dMS) followed by targeted analysis to facilitate this discovery. To trace the non-human dMS identified peptides back to its genetic origin by next generation sequencing (NGS) cDNA libraries were generated using degenerate oligos corresponding to the identified peptides. In a pilot study DPS identified both viral and human biomarkers. Based on the identified peptides that are differentially expressed in the virus positive tumor sample, degenerate oligos were designed. Degenerate oligos or LNA modified degenerate oligos or a modified oligo(dT) SMART primer were used to facilitate reverse transcription from viral or viral and host RNA. NGS analysis revealed seven times more MCV reads in degenerate oligo primed RNAseq samples compared to polyA-based sequencing reads.
Project description:The TAP transporter is responsible for transferring cytosolic peptides into the ER where they can be loaded onto MHC molecules. Deletion of TAP results in a drastic reduction of MHC surface expression and alters the presented peptide pattern. Using the TAP deficient cell line LCL721.174 and its TAP expressing progenitor cell line LCL721.45, we have identified and quantified more than 160 HLA ligands, 50 out of which were presented TAP independently. Peptides which were predominantly presented on the TAP deficient LCL721.174 cell line had a decreased MHC binding affinity according to their SYFPEITHI and BIMAS score. About half of the identified TAP independently presented peptides were not derived from signal sequences and may partly be generated by the proteasome. Furthermore, we have excluded that different HLA presentation ratios were due to varying expression of the respective protein or due to changes in the antigen loading complex. Features of TAP-independently presented peptides as well as proteasomal contribution to their generation provides an insight into basic immunological mechanisms. Keywords: differential mass spectrometry, TAP independent, antigen presentation
Project description:The existence of most peptide segments of CYTB-187AA to high confidence in five cell lines using mass spectrometry and the identification of CYTB-187AA interacting proteins.
Project description:Advances in several key technologies, including MHC peptidomics, has helped fuel our understanding of basic immune regulatory mechanisms and identify T cell receptor targets for the development of immunotherapeutics. Isolating and accurately quantifying MHC-bound peptides from cells and tissues enables characterization of dynamic changes in the ligandome due to cellular perturbations. This multi-step analytical process remains challenging, and throughput and reproducibility are paramount for rapidly characterizing multiple conditions in parallel. Here, we describe a robust and quantitative method whereby peptides derived from MHC-I complexes from a variety of cell lines, including challenging adherent lines, can be enriched in a semi-automated fashion on reusable, dry-storage, customized antibody cartridges. TOMAHAQ, a targeted mass spectrometry technique that combines sample multiplexing and high sensitivity, was employed to characterize neoepitopes displayed on MHC-I by tumor cells and to quantitatively assess the influence of neoantigen expression and induced degradation on neoepitope presentation.