The aim of this study was to evaluate sequence coverage of five model proteins (CYB5A, SMAD4, RAB27B, FECH, and CXXC1) by means of shotgun proteomic data analysis employing different methods of data treatment including database-dependent search engines (MASCOT and X!Tandem) and de novo sequencing software ((PEAKS, Novor, and PepNovo+). In order to achieve maximal results, multiprotease hydrolysis including enzymes trypsin, LYS-C, ASPN and GluC was performed in solution and using the FASP method. High resolution mass spectrometry was carried out with a Q EXACTIVE HF hybrid mass spectrometer in the positive ionization mode; parent ions with the highest intensity and a charge range from +2 to +6 were fragmented in the HCD mode. 27 experiments were carried out (hydrolysis with each of 5 enzymes in solution, 4 for the FASP protocol, three technical repeats). Using parameters limiting false identification of peptides, the search engines and de novo sequencing software gave similar results. The degree of sequence coverage was not at least 40%, and in the best cases it reached 80-90%. The use of de novo sequencing software resulted in identification of the Y12H amino acid substitution in one model protein (CYB5A).
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Keywords: shotgun proteomics, de novo sequencing, mass spectrometry, data processing
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Mikurova A.V., Novikova S.E., Skvortsov V.S., Alekseychuk N.N., Rybina A.V., Miroshnichenko Yu.V. (2017) The sequence coverage in different methods of mass spectrometry data analysis obtained on model proteins. Biomeditsinskaya Khimiya, 63(5), 397-404.
Mikurova A.V. et al. The sequence coverage in different methods of mass spectrometry data analysis obtained on model proteins // Biomeditsinskaya Khimiya. - 2017. - V. 63. -N 5. - P. 397-404.
Mikurova A.V. et al., "The sequence coverage in different methods of mass spectrometry data analysis obtained on model proteins." Biomeditsinskaya Khimiya 63.5 (2017): 397-404.
Mikurova, A. V., Novikova, S. E., Skvortsov, V. S., Alekseychuk, N. N., Rybina, A. V., Miroshnichenko, Yu. V. (2017). The sequence coverage in different methods of mass spectrometry data analysis obtained on model proteins. Biomeditsinskaya Khimiya, 63(5), 397-404.
Ni W., Lin M., Salinas P., Savickas P., Wu S.L., Karger B.L. (2013) J. Am. Soc. Mass Spectrometry, 24(1), 125-133. CrossRef Scholar google search
Ershov P., Mezentsev Y., Gnedenko O., Mukha D., Yantsevich A., Britikov V., Kaluzhskiy L., Yablokov E., Molnar A., Ivanov A., Lisitsa A., Gilep A., Usanov S. (2012) Proteomics, 12(22), 3295-3298. CrossRef Scholar google search
Vaudel M., Barsnes H., Berven F.S., Sickmann A., Martens L. (2011) Proteomics, 11(5), 996-999. CrossRef Scholar google search
Zhang J., Xin L., Shan B., Chen W., Xie M., Yuen D., Zhang W., Zhang Z., Lajoie G.A., Ma B. (2012) Mol. Cell. Proteomics, 11(4), M111-010587. CrossRef Scholar google search
Muth T., Weilnböck L., Rapp E., Huber C.G., Martens L., Vaudel M., Barsnes H. (2014) J. Proteome Res., 13(2), 1143-1146. CrossRef Scholar google search
