Dataset Information

MudPIT analysis of HSV-1 infected and mock-infected microsomal membranes and nuclear envelopes

ABSTRACT: Nuclear envelopes (NEs) and microsomal membranes (MMs) were isolated from HeLa cells infected or not with HSV-1 using well-established procedures as described in: Wilkie GS, et al. (2011) Mol Cell Proteomics 10: M110 003129. Korfali N, et al. (2010) Mol Cell Proteomics 9: 2571-2585. Florens L, Korfali N, Schirmer EC (2008) Methods Mol Biol 432: 117-137. Membrane pellets were denatured, reduced, alkylated then digested with endoproteinase LysC followed by trypsin. Peptides were analyzed by Multidimensional Protein Identification Technology (MudPIT) as described in: Florens L, Washburn MP (2006) Methods Mol Biol 328: 159-175. RAW files were extracted into ms2 file format using RawDistiller v. 1.0 as in: Zhang, Y.; Wen, Z.; Washburn, M. P.; Florens, L. (2011) Anal Chem, 83, (24), 9344-51. MS/MS spectra were queried for peptide sequence information using SEQUEST v.27 (rev.9) as in: Eng J, McCormack A, Yates Jr (1994) J Am Soc Mass Spectrom 5: 976-989. Results from different runs were compared using DTASelect and CONTRAST as in: Tabb DL, McDonald WH, Yates JR (2002) J Proteome Res 1: 21-26. To estimate relative protein levels, distributed normalized spectral abundance factors (dNSAFs) were calculated for each non-redundant protein as in: Zhang Y, Wen Z, Washburn MP, Florens L (2010) Anal Chem 82: 2272-2281.

INSTRUMENT(S): LTQ

ORGANISM(S): Homo Sapiens (ncbitaxon:9606) Herpes Simplex Virus (type 1 / Strain 17) (ncbitaxon:10299)

SUBMITTER: Laurence Florens

PROVIDER: MSV000079886 | MassIVE | Tue Jul 05 07:55:00 BST 2016

SECONDARY ACCESSION(S): PXD004519

REPOSITORIES: MassIVE

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Publications

Host Vesicle Fusion Protein VAPB Contributes to the Nuclear Egress Stage of Herpes Simplex Virus Type-1 (HSV-1) Replication.

Saiz-Ros Natalia N Czapiewski Rafal R Epifano Ilaria I Stevenson Andrew A Swanson Selene K SK Dixon Charles R CR Zamora Dario B DB McElwee Marion M Vijayakrishnan Swetha S Richardson Christine A CA Dong Li L Kelly David A DA Pytowski Lior L Goldberg Martin W MW Florens Laurence L Graham Sheila V SV Schirmer Eric C EC

Cells 20190203 2

The primary envelopment/de-envelopment of Herpes viruses during nuclear exit is poorly understood. In Herpes simplex virus type-1 (HSV-1), proteins pUL31 and pUL34 are critical, while pUS3 and some others contribute; however, efficient membrane fusion may require additional host proteins. We postulated that vesicle fusion proteins present in the nuclear envelope might facilitate primary envelopment and/or de-envelopment fusion with the outer nuclear membrane. Indeed, a subpopulation of vesicle-a ...[more]

PMID: 30717447

Similar Datasets

Project description:Plasmids encoding full-length open-reading frames (ORF) of heterogeneous nuclear ribonucleoproteins fused to a HaloTag (HT) were transfected in human HEK293T cells in biological duplicates. The expressed proteins were: HNRNPA0 (NM_006805; NP_006796.1), HNRNPA1 (NM_002136; NP_002127.1), HNRNPD (NM_002138; NP_002129.2), HNRNPF (NM_001098206; NP_001091676.1), HNRNPH1 (NM_005520; NP_005511.1), HNRNPK (NM_001318187; NP_001305116.1), HNRNPM (NM_005968; NP_005959.2), HNRNPR (NM_005826; NP_005817.1), HNRNPU (NM_031844; NP_114032.2), HNRNPUL1 (NM_007040; NP_008971.2), RBFOX2 (NM_001082578; NP_001076047.1), and UPF1 (NM_002911; NP_002902.2). Cells were lysed 24 hours post-transfection, and half of the lysates were left untreated, while the remaining half were subjected to stringent RNase digestion (2ul RNAse A; A797C 4mg/ml for each 12 million cell pellet lysate). Protein complexes were covalently captured on an HT affinity resin and interacting proteins were eluted and purified for mass spectrometry as described (Daniels, D.L., et al., (2012) J. Proteome Res., 11(2):564-75). TCA-precipitated roteins were digested with endoproteinase LysC followed by trypsin. The resulting peptide mixtures were analyzed by Multidimensional Protein Identification Technology (MudPIT) on an LTQ ion trap mass spectrometer as described (Florens L, Washburn MP. (2006) Methods Mol. Biol., 328:159-75). RAW files were extracted into .ms2 file format using RawDistiller v. 1.0 (Zhang, Y., Wen, Z., Washburn, M. P., and Florens, L. (2011) Anal. Chem. 83, 9344-9351.). The MS/MS spectra were searched using SEQUEST v.27 rev.9 (Eng, McCormack, and Yates (1994) J. Amer. Mass Spectrom. 5, 976-989.) with a peptide mass tolerance of 3 amu and of +/- 0.5 amu for fragment ions. To account for alkylation by chloroacetamide (CAM), 57.02146 Da were added statically to cysteine residues for all searches. No enzyme specificity was imposed during the SEQUEST searches against a protein database containing 29375 non-redundant Homo sapiens proteins (NCBI 2010-11-22 release), as well as 163 usual contaminants such as human keratins, IgGs and proteolytic enzymes. To estimate false discovery rates (FDR), each protein sequence was randomized (keeping the same amino acid composition and length) and the resulting "shuffled" sequences were added to the database used for the SEQUEST searches, for a total search space of 59076 amino acid sequences. Spectra/peptide matches (PSMs) were filtered using conservative criteria using DTASelect (Tabb, McDonald, and Yates (2002) J. Proteome Res. 1, 21-26). PSMs were only retained if they had a DeltCn of at least 0.08. Minimum XCorr values were set at 1.8 for singly-, 2.0 for doubly-, and 3.0 for triply-charged spectra. In addition, peptides had to be at least 7 amino acids long and fully tryptic. Compressed and archived directories containing the complete mass spectrometry dataset for each anlayzed sample: mass spectrometry files (.raw), peak files (.ms2), SEQUEST search files (sequest.params and .sqt), as well as DTASelect result files (DTASelect.params and DTASelect.txt/html). Use "tar -xzf" command in Linux to restore folders for each sample and files therein.

Project description:MudPIT analyses of novel spliceosomal SF3B component in SAGA complex Stable Drosophila S2 cell lines expressing SF3B5 (CG11985, NP_652189.1), U2B (SNF; CG4528, NP_511045.1), Ada2b-PB (CG9638, NP_001027151.1), Spt3 (CG3169, NP_650146.1), Spt20 (CG17689, NP_648659.2), ATXN7 (CG9866, NP_722803.1), Ada1 (CG31866, NP_723703.1), SAF6 (CG3883, NP_608545.1), Sgf29 (CG30390, NP_726051.1), and WDA (CG4448, NP_651136.1) in the pRmHa3-CHA2FL2 vector were generated as described (Guelman S, et al., Mol Cell Biol. 2006;26:7178-89). Protein complexes were purified by tandem FLAG-HA affinity purification as described (Weake VM, et al. Genes Dev. 2009;23:2818-23). Two negative controls were purified in parallel from S2 cells without any vectors and from S2 cells expressing CG6459. Where indicated, 250 ug/mL RNAseA was added to soluble nuclear extract from WDA-expressing cells prior to immunoprecipitation. Proteins were digested with endoproteinase LysC followed by trypsin. The resulting peptide mixtures were analyzed by Multidimensional Protein Identification Technology (MudPIT) on an LTQ ion trap mass spectrometer as described (Florens L, Washburn MP. Methods Mol Biol 2006;328:159-75). RAW files were extracted into .ms2 file format using RawDistiller v. 1.0 (in-house developed software). The MS/MS spectra were searched using SEQUEST v.27 (rev.9) with a peptide mass tolerance of 3 amu and of +/- 0.5 amu for fragment ions. No enzyme specificity was imposed during the SEQUEST searches against a protein database containing 18425 non-redundant Drosophila melanogaster proteins (NCBI 2011-04-11 release), as well as 177 usual contaminants such as human keratins, IgGs and proteolytic enzymes. To estimate false discovery rates (FDR), each protein sequence was randomized (keeping the same amino acid composition and length) and the resulting "shuffled" sequences were added to the database used for the SEQUEST searches, for a total search space of 37204 amino acid sequences. To account for alkylation by CAM, 57.02146 Da were added statically to cysteine residues for all searches.