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Probing the Function of Alternative Proteins in Cell Reprogramming by Large Scale Proteomics and Cross - Linking Mass Spectrometry

ABSTRACT: Many proteins are lacking from reference databases because they are issued from alternative ORFs neither respecting the rules decreed for the genome annotation or for the protein translation from mRNA. It was evidenced that not only a single protein is therefore translated from an mRNA. However, the function of these Alternative Proteins (AltProts) remain largely unknown. Here, we were interested by looking to the function of AltProts in the context of cancer cell reprogramming. We have developed a large scale approach based on shot-gun proteomics and cross-linking mass spectrometry (XL-MS) to identify the regulation of the reference proteins (RefProts) versus the AltProts and find the their interaction partners. The study was conducted from NCH82 human glioma cells which were stimulated by the protein kinase A (PKA) activator Forskolin upon 16H, 24H and 48H to induce cell differentiation and epithelial-mesenchymal transition (EMT). The data have shown to enable tracing back the function of the AltProts by combining experimental data to in silico analysis using cytoscape with ClueGo for GO Term annotation and enrichment of pathways with String. Very interestingly many AltProts demonstrate to be involved in the regulation of tRNA through their interaction with aaRS proteins and of the cellular mobility.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Tristan Cardon

LAB HEAD: Michel Salzet

PROVIDER: PXD014642 | Pride | 2021-02-15

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	HS_GRCh38_altorf_20170421.fasta	Fasta
	MSMS_File_Nuclei.zip	Other
	MSMS_File_TimeCourse.zip	Other
	MSMS_File_TotalCell.zip	Other
	RAW_File_Nuclei.zip	Other

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Project description:In this study we analyzed and compared total proteome and secretome of the wild-type and JN1 mutant strains of Bordetella pertussis. The single-nucleotide transversion in the 5’-UTR of the rplN gene of JN1 mutant led to the increased transcription of the whole operon encoding ribosomal proteins and of the adjoining rpoA gene. These events led to the downregulation and decreased secretion of virulence factors on the background ofgenerally deregulated expression of B. pertussis genome. To get deeper inside in the molecular mechanisms of the observed genome deregulation we then performed the immunoprecipitation of RpoA and compared its binding partners in wild-type and JN1 mutant strains. Nano Reversed phase column (EASY-Spray column, 50 cm x 75 µm ID, PepMap C18, 2µm particles, 100Å pore size) was used for LC/MS analysis. Mobile phase buffer A was composed of water and 0.1% formic acid. Mobile phase B was composed of acetonitrile and 0.1% formic acid. Samples were loaded onto the trap column (Acclaim PepMap300, C18, 5µm, 300Å wide Pore, 300 µm x 5 mm) at a flow rate of 15 μl/min. Loading buffer was composed of water, 2% acetonitrile and 0.1% trifluoroacetic acid. Peptides were eluted with gradient of B from 4% to 35% over 60 min at a flow rate of 300 nl/min. Eluting peptide cations were converted to gas-phase ions by electrospray ionization and analyzed on a Thermo Orbitrap Fusion (Q-OT- qIT, Thermo). Survey scans of peptide precursors from 350 to 1400 m/z were performed at 120K resolution (at 200 m/z) with a 5 × 105 ion count target. Tandem MS was performed by isolation at 1.5 Th with the quadrupole, HCD fragmentation with normalized collision energy of 30, and rapid scan MS analysis in the ion trap. The MS 2 ion count target was set to 104 and the max injection time was 35 ms. Only those precursors with the charge state 2–6 were sampled for MS 2. The dynamic exclusion duration was set to 45 s with a 10ppm tolerance around the selected precursor and its isotopes. Monoisotopic precursor selection was turned on. The instrument was run in top speed mode with 2s cycles (76). The data were analyzed and quantified with label-free quantification (LFQ) algorithms in MaxQuant v1.6.3.3 (77) and the Andromeda search engine(78). The false discovery rate (FDR) parameter was set to 1 % for both proteins and peptides. The enzyme specificity of trypsin was set as C-terminal to Arg and Lys. Carbamidomethylation was set as the fixed modification, while N-terminal protein acetylation and methionine oxidation were variable modifications. Maximal number of missed cleavages was set to 2. All hits identified in searches as contaminants were filtered out. The data were searched against Bordetella pertussis reference proteome database (strain Tohama I / ATCC BAA-589 / NCTC 13251).

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Probing the Function of Alternative Proteins in Cell Reprogramming by Large Scale Proteomics and Cross - Linking Mass Spectrometry

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