MassIVE MSV000079669

Partial Public PXD004000

MudPIT analyses of the proteins co-purified with Halo-tagged Human HNRNP proteins from HEK293T cells with and without RNAse

Subscribe Comment Convert Spectra Add Reanalysis

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. [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Halo Tag ; MudPIT ; heterogeneous nuclear ribonucleoprotein ; RNA binding protein

Contact

Principal Investigators:
(in alphabetical order) 		Laurence Florens
Submitting User: laflorens

Publications

Brannan, K.W., Jin, W., Huelga, S.C., Banks, C.A.S., Gilmore, J.M., Florens, L., Washburn, M.P., Van Nostrand, E.L., Pratt, G.A., Schwinn, M.K., Daniels, D., Yeo, G. W.
SONAR discovers RNA binding proteins from analysis of large-scale protein-protein interactomes.
Mol Cell. 2016 Oct 20;64(2):282-293. doi: 10.1016/j.molcel.2016.09.003. Epub 2016 Oct 6.

Number of Files:
Total Size:
Spectra:
Subscribers:
 
Owner Reanalyses
Experimental Design
    Conditions:
    Biological Replicates:
    Technical Replicates:
 
Identification Results
    Proteins (Human, Remapped):
    Proteins (Reported):
    Peptides:
    Variant Peptides:
    PSMs:
 
Quantification Results
    Differential Proteins:
    Quantified Proteins:
 
Browse Dataset Files
 
FTP Download Link (click to copy):

Species

Instrument

Modifications

- Dataset Reanalyses

+ Dataset History

Click here to queue conversion of this dataset's submitted spectrum files to open formats (e.g. mzML). This process may take some time.

When complete, the converted files will be available in the "ccms_peak" subdirectory of the dataset's FTP space (accessible via the "FTP Download" link to the right).
Number of distinct conditions across all analyses (original submission and reanalyses) associated with this dataset.

Distinct condition labels are counted across all files submitted in the "Metadata" category having a "Condition" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct biological replicates across all analyses (original submission and reanalyses) associated with this dataset.

Distinct replicate labels are counted across all files submitted in the "Metadata" category having a "BioReplicate" or "Replicate" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct technical replicates across all analyses (original submission and reanalyses) associated with this dataset.

The technical replicate count is defined as the maximum number of times any one distinct combination of condition and biological replicate was analyzed across all files submitted in the "Metadata" category. In the case of fractionated experiments, only the first fraction is considered.

"N/A" means no results of this type were submitted.
Originally identified proteins that were automatically remapped by MassIVE to proteins in the SwissProt human reference database.

"N/A" means no results of this type were submitted.
Number of distinct protein accessions reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct unmodified peptide sequences reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct peptide sequences (including modified variants or peptidoforms) reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Total number of peptide-spectrum matches (i.e. spectrum identifications) reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct proteins quantified across all analyses (original submission and reanalyses) associated with this dataset.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct proteins found to be differentially abundant in at least one comparison across all analyses (original submission and reanalyses) associated with this dataset.

A protein is differentially abundant if its change in abundance across conditions is found to be statistically significant with an adjusted p-value <= 0.05 and lists no issues associated with statistical tests for differential abundance.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

"N/A" means no results of this type were submitted.
This dataset may not contain all raw spectra data as originally deposited in PRIDE. It has been imported to MassIVE for reanalysis purposes, so its spectra data here may consist solely of processed peak lists suitable for reanalysis with most software.