Gerben.menschaert

Use of HLA peptidomics and whole exome sequencing to identify human immunogenic neo-antigens.

Oncotarget. 2016 Jan 20;

Authors: Kalaora S, Barnea E, Merhavi-Shoham E, Qutob N, Teer JK, Shimony N, Schachter J, Rosenberg SA, Besser MJ, Admon A, Samuels Y

Abstract
The antigenicity of cells is demarcated by the peptides bound by their Human Leucocyte Antigen (HLA) molecules. Through this antigen presentation, T cell specificity response is controlled. As a fraction of the expressed mutated peptides is presented on the HLA, these neo-epitopes could be immunogenic. Such neo-antigens have recently been identified through screening for predicted mutated peptides, using synthetic peptides or ones expressed from minigenes, combined with screening of patient tumor-infiltrating lymphocytes (TILs). Here we present a time and cost-effective method that combines whole-exome sequencing analysis with HLA peptidome mass spectrometry, to identify neo-antigens in a melanoma patient. Of the 1,019 amino acid changes identified through exome sequencing, two were confirmed by mass spectrometry to be presented by the cells. We then synthesized peptides and evaluated the two mutated neo-antigens for reactivity with autologous bulk TILs, and found that one yielded mutant-specific T-cell response. Our results demonstrate that this method can be used for immune response prediction and promise to provide an alternative approach for identifying immunogenic neo-epitopes in cancer.

PMID: 26819371 [PubMed - as supplied by publisher]

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Comparison of two phenotypically distinct lattice corneal dystrophies caused by mutations in the transforming growth factor beta induced (TGFBI) gene.

Proteomics Clin Appl. 2014 Apr;8(3-4):168-77

Authors: Poulsen ET, Runager K, Risør MW, Dyrlund TF, Scavenius C, Karring H, Praetorius J, Vorum H, Otzen DE, Klintworth GK, Enghild JJ

Abstract
PURPOSE: In this study, we investigated whether the phenotypic difference observed between two lattice corneal dystrophy type 1 (LCD type 1) cases caused by either a single A546D substitution or an A546D/P551Q double substitution in TGFBIp (transforming growth factor beta induced protein) can be ascribed to (i) a difference in the proteomes of corneal amyloid deposits, (ii) altered proteolysis of TGFBIp, or (iii) structural changes of TGFBIp introduced by the P551Q amino acid substitution.
EXPERIMENTAL DESIGN: Amyloid deposits were isolated from the corneas of two siblings with LCD type 1 resulting from A546D/P551Q mutations in the TGFBI gene using laser capture microdissection and subsequently analyzed by LC-MS/MS. Proteolytic processing of TGFBIp was addressed by counting peptide spectra. Lastly, to study the possible effect of the P551Q substitution, recombinant FAS1-4 domain variants were subjected to in vitro stability assays.
RESULTS: The amyloid proteomes and TGFBIp processing of the two A546D/P551Q LCD type 1 cases were similar to each other as well as to the A546D amyloid proteome previously reported by us. The stability assays revealed a minor destabilization of the FAS1-4 domain upon the addition of the P551Q mutation, moreover, it resulted in different accessibility to tryptic cleavage sites between the A546D and A546D/P551Q mutant FAS1-4 domain variants.
CONCLUSION AND CLINICAL RELEVANCE: The difference in A546D and A546D/P551Q LCD type 1 phenotypes cannot be ascribed to altered corneal amyloid composition or altered in vivo proteolytic processing of TGFBIp. Instead, a small difference in thermodynamic stability introduced by the P551Q mutation most likely causes structural changes of TGFBIp. The MS proteomics data have been deposited to the ProteomeXchange with identifier PXD000307 (http://proteomecentral.proteomexchange.org/dataset/PXD000307).

PMID: 24302499 [PubMed - indexed for MEDLINE]

Functionalized magnetic nanoparticles for sample preparation in proteomics and peptidomics analysis.

Chem Soc Rev. 2013 Aug 9;

Authors: Li Y, Zhang X, Deng C

Abstract
Sample preparation is a fundamental step in the proteomics and peptidomics workflow. Due to their good biocompatibility, superparamagnetic property, and high binding capacity, magnetic nanoparticles (MNPs) functionalized with different active moieties have been widely applied in recent years in various sample preparation procedures in proteomics and peptidomics analysis. The magnetic cores of the MNPs facilitate elegant handling using only magnetic devices and their small diameters are advantageous for increasing the sensitivity when using subsequent mass spectrometry (MS) analysis or gel electrophoresis. This review mainly focuses on overviewing present advances in the preparation and application of functionalized magnetic nanoparticles for sample preparation in proteomics and peptidomics analysis, including protein digestion, enrichment of low-abundance peptides/proteins and specific enrichment of peptides/proteins with post-translational modifications, such as phosphorylation and glycosylation.

PMID: 23933677 [PubMed - as supplied by publisher]

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ESCAPE: database for integrating high-content published data collected from human and mouse embryonic stem cells.

Database (Oxford). 2013;2013(0):bat045

Authors: Xu H, Baroukh C, Dannenfelser R, Chen EY, Tan CM, Kou Y, Kim YE, Lemischka IR, Ma'ayan A

Abstract
High content studies that profile mouse and human embryonic stem cells (m/hESCs) using various genome-wide technologies such as transcriptomics and proteomics are constantly being published. However, efforts to integrate such data to obtain a global view of the molecular circuitry in m/hESCs are lagging behind. Here, we present an m/hESC-centered database called Embryonic Stem Cell Atlas from Pluripotency Evidence integrating data from many recent diverse high-throughput studies including chromatin immunoprecipitation followed by deep sequencing, genome-wide inhibitory RNA screens, gene expression microarrays or RNA-seq after knockdown (KD) or overexpression of critical factors, immunoprecipitation followed by mass spectrometry proteomics and phosphoproteomics. The database provides web-based interactive search and visualization tools that can be used to build subnetworks and to identify known and novel regulatory interactions across various regulatory layers. The web-interface also includes tools to predict the effects of combinatorial KDs by additive effects controlled by sliders, or through simulation software implemented in MATLAB. Overall, the Embryonic Stem Cell Atlas from Pluripotency Evidence database is a comprehensive resource for the stem cell systems biology community. Database URL: http://www.maayanlab.net/ESCAPE.

PMID: 23794736 [PubMed - in process]

Zm908p11, encoded by a short open reading frame (sORF) gene, functions in pollen tube growth as a profilin ligand in maize.

J Exp Bot. 2013 May;64(8):2359-72

Authors: Dong X, Wang D, Liu P, Li C, Zhao Q, Zhu D, Yu J

Abstract
Double fertilization of flowering plants depends on the targeted transportation of sperm to the embryo sac by the pollen tube. Currently, little is known about the underlying molecular mechanisms that regulate pollen germination and pollen tube growth in maize (Zea mays). Here, a maize pollen-predominant gene Zm908, with several putative short open reading frames (sORFs), was isolated and characterized. The longest ORF of Zm908 encodes a small protein of 97 amino acids. This was designated as Zm908p11 and is distributed throughout the maize pollen tube. Western blot detected the small peptide in mature pollen. Quantitative reverse transcription-PCR and northern blot analysis revealed that Zm908p11 was expressed predominantly in mature pollen grains. Ectopic overexpression of full-length Zm908 and Zm908p11 in tobacco resulted in defective pollen, while transgenic tobacco plants with a site-specific mutation or a frameshift mutation of Zm908p11 showed normal pollen development. Overexpression of Zm908p11 in maize decreased pollen germination efficiency. Maize pollen cDNA library screening and protein-protein interaction assays demonstrated that Zm908p11 interacts with maize profilin 1 (ZmPRO1). A microarray analysis identified 273 up-regulated and 203 down-regulated genes in the overexpressing transgenic Zm908p11 pollen. Taken together, these results indicate that Zm908 functions as Zm908p11, and binds to profilins as a novel ligand, with a required role during pollen tube growth in maize. Accordingly, a model is proposed for the role of Zm908p11 during pollen tube growth in maize.

PMID: 23676884 [PubMed - in process]