Dataset Information

Proteomic identification of monoclonal antibodies from rabbit serum and analysis of FDR using differential cysteine labeling

ABSTRACT: Proteomic analysis of serum antibodies is challenging due to the non-germline origin of immunoglobulin (Ig) sequences and the complexity of Ig primary structure, with regions of hypervariable sequence interspersed within highly conserved framework sections resulting in an unusually high frequency of peptide-spectrum mis-identification using standard reference database search methods. Using a strategy of differential cysteine labeling with two alkylating reagents, we examined peptide-spectrum matches and decoy-based error modeling for the interpretation of antibody-derived mass spectral data.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Oryctolagus Cuniculus (rabbit)

TISSUE(S): Blood Serum

SUBMITTER: Daniel Boutz

LAB HEAD: Edward Marcotte

PROVIDER: PXD000916 | Pride | 2014-04-24

REPOSITORIES: Pride

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Publications

Proteomic identification of monoclonal antibodies from serum.

Boutz Daniel R DR Horton Andrew P AP Wine Yariv Y Lavinder Jason J JJ Georgiou George G Marcotte Edward M EM

Analytical chemistry 20140501 10

Characterizing the in vivo dynamics of the polyclonal antibody repertoire in serum, such as that which might arise in response to stimulation with an antigen, is difficult due to the presence of many highly similar immunoglobulin proteins, each specified by distinct B lymphocytes. These challenges have precluded the use of conventional mass spectrometry for antibody identification based on peptide mass spectral matches to a genomic reference database. Recently, progress has been made using botto ...[more]

PMID: 24684310

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Project description:Our computational analyses of sequencing depth and the discovery rates of sequence elements in the 3M-bM-^@M-^YUTR strongly demonstrate that interrogation of the 3M-bM-^@M-^YUTRome in specific tissues and cell types across development will greatly expand the identification of new 3M-bM-^@M-^YUTR isoforms and the sequence elements therein. Therefore, we will generate and sequence the 3M-bM-^@M-^YUTRs from the cell types isolated from the developing germline, mature germ cells, and early embryogenesis. In addition, we will identify the 3M-bM-^@M-^YUTRs for the transcripts isolated from the major somatic tissues during late- and post-embryonic development. Based on our sequencing estimates, we anticipate that the tissue-specific interrogation of the 3M-bM-^@M-^YUTRome will reveal a far greater diversity of novel 3M-bM-^@M-^YUTR isoform expression that is masked in the whole-worm 3M-bM-^@M-^YUTRome sequence data. Using the transgenic myo-3::PABP strain, we have generated polyA-captured libraries for late embryos and across the major stages of post-embryonic development for the muscle transcriptome. We propose to generate these polyA-captured libraries for transcripts expressed in the other major tissues across development. PolyA-captured libraries will be generated for deep sequencing following the tissue-specific isolation of mRNAs by PABP pulldowns (the PABP immunoprecipitations will take advantage of a FLAG epitope which is fused to PABP in all of the transgenic strains). We propose to validate the expression of tissue- specific transcripts by qPCR for select transcripts known to be expressed in those particular tissues. In addition, following the strategy we have employed for the large-scale polyA-captured libraries from muscle, we will perform quality checks for 3M-JM-< end capture by manually sequencing ~60 clones isolated from each library. Once we have obtained the deep sequencing data, we will analyze all of the sequence reads using the bioinformatics pipeline that we established for the whole-worm polyA- captured sequences (Mangone et al., 2010). Four samples representing gravid, L2, L3, and L4 developmental stages were analyzed.

Dataset Information

Proteomic identification of monoclonal antibodies from rabbit serum and analysis of FDR using differential cysteine labeling

Publications

Proteomic identification of monoclonal antibodies from serum.

Similar Datasets

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