ABSTRACT: We performed an in-depth quantitative mass spectrometry-based analysis to probe dynamic proteome changes during somatic cell reprogramming. We analyzed the precursor secondary embryonic mouse fibroblasts (MEFs), 6 intermediate time points (day 2, 5, 8, 11, 16 and 18) at high-dox concentrations, 3 intermediate time points collected after modulating transgene expression, the secondary and primary iPSCs and, as a standard of pluripotency, Rosa26-rtTA transgenic embryonic stem cells. Our data revealed defined waves of proteome resetting, with the first wave occurring 48 hours after transgene expression, involving specific biological processes. A second wave of proteome reorganization was evident in a later stage of reprogramming, where we could distinguish and characterize two distinct pluripotent cellular populations. The proteomes of these cell states are clearly distinct, with altered levels of proteins involved in cell adhesion, epigenetic mechanisms, cell proliferation and pluripotency. Our proteome resource can be used to identify the molecular players involved in key steps during reprogramming, gaining knowledge to improve the fidelity of reprogramming and advance our knowledge of cellular plasticity. Data analysis: MS raw data were processed with Proteome Discoverer (version 1.3, Thermo Electron). Basically, peptide identification was performed with Mascot 2.3 (Matrix Science) against a concatenated forward-decoy UniPROT database supplemented with all the frequently observed contaminants in MS (version 5.62). The following parameters were used: 50 ppm. precursor mass tolerance, 0.02 Da fragment ion tolerance, up to 2 missed cleavages, carbamidomethyl cysteine as fixed modification, oxidized methionine and TMT modification on N-Term and Lysine as variable modifications. Finally, we performed a deconvolution of the high resolution MS2 spectra, by which all the fragment ions isotopic distributions were converted to an m/z value corresponding to the monoisotopic single charge. Reporter ion based quantification method was chosen in Proteome Discoverer, with the following requirements for reporter ion integration in the MS2 spectra; mass accuracy of maximum 20 p.p.m., peptide ratio maximum limit 100. In order to minimize ratio distortion due to the presence of more than one peptide species within the precursor ion isolation width, we also reject the quantification of MS/MS spectra having a co-isolation higher than 30%. Finally, results were filtered using the following criteria: (i) mass deviations of +-5 p.p.m., (ii) Mascot Ion Score of at least 25, (iii) a minimum of 7 amino-acid residues per peptide and (iv) position rank 1 in Mascot search. As a result, we obtained peptide FDRs below 1% for all of the three peptide mixtures analyzed.
Marco Benevento 