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Summary

Title

Colon cancer stem cell over-expressing ΔNp63α

Description

p63, a member of p53 family, is transcribed in different variants, containing (TA) or lacking (ΔN) the N-terminal transactivation domain. Although the proteins of p53 family share high sequence and structural similarities, distinct functions for p63 are emerging. Here we provided a quantitative proteomic analysis by stable isotope dimethyl labeling of colon cancer stem cells over-expressing ΔNp63α in order to investigate the cellular pathways modulated by this p63 isoform.

Sample Processing Protocol

Protein extracts from control cells and cells over-expressing ΔNp63α were digested by standard protocols and Tryptic peptides were subjected to chemical labeling. 1.5μl of formaldehyde-D0 (20% in water) were mixed to the peptides mixture, vortexed and incubated for 5min at RT. Then 2.5μl of freshly prepared sodium cyanoborohydride (1M) were added and allowed to react for 1h at RT. Deuterium labeling was performed by similar procedure, but by using formaldehyde-D2 (20% in water). Light and heavy labeled samples were mixed 1:1 prior peptides separation by isoelectric focusing. We analyzedNp63- D0 with CTRL- D2 (1:1) and the inverted labeled samples Np63- D2 with CTRL- D0 (1:1). Immobilized pH Gradient Isoelectric Focusing (IPG-IEF) was used for separation of labeled peptides. Focused strip were cut in 17 pieces and peptides were extracted. Five microliters of each sample were separated by a Proxeon Easy-nLC II (Thermo Scientific,Waltham, MA) chromatographic system equipped with an EASY-Column C18. 5 μm, 100 μm×20mm precolumn (Thermo Scientific) and using an Acclaim PepMap100 C18, 5 μm, 75 μm×25mm(Dionex, Thermo Scientific) nanoscale LC column. Mobile phase A was water with 0.1% FA and mobile phase B was 0.1% FA in acetonitrile. Peptides were separated with a reverse phase gradient of 5–35% mobile phase B over 57 min at flow rate of 300 nl/min, a rinse with 100% mobile phase B for 10 min. The chromatographic system was coupled online with a MicrOTOF Q-II mass spectrometer (Bruker-Daltonics, Bremen, Germany), equipped with an ESI nano Sprayer ion source. Spectra acquisition in the mass range 400-1400m/z, was performed by using an Auto (MS/MS) mode method optimized for quantitative proteomics (Bruker-Daltonics, Bremen, Germany), with number of precursor ions set to 5, MS spectra rate of 1.0 Hz, using Argon in collision gas cell, and the SILE option selected, applying a delta mass of 4.0251m/z fixed for peptides pairs in the SILE option.

Data Processing Protocol

Spectra were processed in Multifiles Projects by using Mascot Distiller v2.4.3.3, applying the default processing methods optimized for Q-TOF instrument. Peptide identification was achieved by in house version of Mascot algorithm (version 2.4), interrogating Swiss-Prot database (released version 2013_02) restricted to Homo sapiens taxonomy (20248 sequences). Search parameters were: carbamidomethylation of cysteines as fixed modification, oxidation of methionines as variable modification, Dimethylation as Quantitation method, one missing cleavage allowed on tryptic peptides, 20ppm for peptide tolerance, 0.1Da for fragment tolerance, p-value< 0.05 for peptide significant value of identification, Decoy option active. Proteins identified with one unique peptide were manually validated. After protein identification only peptides matching the following criteria were included in the final Quantitation analysis: p-value< 0.05 for peptide significant value, bold red, unique sequences, standard error less than 0.2, correlation threshold above 0.9, fraction threshold al least of 0.5.

Contact

Simona D'Aguanno, Experimental Chemotherapy Laboratory Regina Elena National Cancer Institute, Rome, Italy
Prof. Andrea Urbani, Department of Experimental Medicine, University of Tor Vergata, Rome, Italy ( lab head )

Submission Date

20/02/2014

Publication Date

17/03/2014

Publication

    D'Aguanno S, Barcaroli D, Rossi C, Zucchelli M, Cortese C, De Cola A, Volpe S, D'Agostino D, Todaro M, Stassi G, Di Ilio C, Urbani A, De Laurenzi V. p63 Isoforms Regulate Metabolism of Cancer Stem Cells. J Proteome Res. 2014 Mar 5 PubMed: 24597989

Assay

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Showing 1 - 10 of 27 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 35059 DNp63 Exp2 fract1 33 58 41 1411 44
2 35057 DNp63 Exp2 fract 10 51 68 56 577 65
3 35033 DNp63 Exp2 fract 12 27 32 27 632 30
4 35058 DNp63 Exp2 fract 13 14 18 14 771 18
5 35034 DNp63 Exp2 fract 14 82 153 104 1134 123
6 35036 DNp63 Exp2 fract 15 13 19 16 937 19
7 35056 DNp63 Exp2 fract 16 102 192 130 1161 159
8 35035 DNp63 Exp2 fract 17 63 109 75 968 92
9 35055 DNp63 Exp 2 fract 2 28 46 28 805 37
10 35038 DNp63 Exp3 Fract 4 120 194 146 1217 181