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. 2017 Oct;63(5):397-404.
doi: 10.18097/PBMC20176305397.

[The sequence coverage in different methods of mass spectrometry data analysis obtained on model proteins]

[Article in Russian]
A V Mikurova  1 , S E Novikova  1 , V S Skvortsov  1 , N N Alekseychuk  1 , A V Rybina  1 , Yu V Miroshnichenko  1
Affiliations

[The sequence coverage in different methods of mass spectrometry data analysis obtained on model proteins]

[Article in Russian]
A V Mikurova et al. Biomed Khim. 2017 Oct.

Abstract
in English, Russian

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).

V rabote provedena otsenka na 5 model'nykh belkakh (CYB5A, SMAD4, RAB27B, FECH i CXXC1) stepeni pokrytiia aminokislotnoĭ posledovatel'nosti v panoramnoĭ proteomike pri ispol'zovanii razlichnykh metodov obrabotki dannykh, vkliuchaiushchikh poiskovye sistemy (Mascot i X!Tandem) i programmy de novo sekvenirovaniia (PEAKS, Novor i PepNovo+). Dlia dostizheniia maksimal'nogo rezul'tata v rabote primenialsia mul'tiproteaznyĭ gidroliz (fermenty Tripsin, Lys-S, AspN i GluC) v rastvore i po metodike FASP. Mass-spektrometricheskiĭ analiz vysokogo razresheniia provodili s pomoshch'iu gibridnogo mass-spektrometra Q Exactive HF v rezhime polozhitel'noĭ ionizatsii, roditel'skie iony s naibol'sheĭ intensivnost'iu i zariadom v diapazone ot +2 do +6, fragmentirovali v rezhime HCD. Vsego bylo provedeno 27 éksperimentov (gidroliz kazhdym iz 5 fermentov v rastvore; 4 dlia FASP-protokola, po tri tekhnicheskikh povtora). Pri ispol'zovanii parametrov, ogranichivaiushchikh lozhnye identifikatsii peptidov, poiskovye sistemy i programmy de novo sekvenirovaniia daiut skhodnye rezul'taty. Stepen' pokrytiia aminokislotnoĭ posledovatel'nosti ne men'she 40%, a v luchshikh sluchaiakh 80-90%. Ispol'zovanie programm de novo sekvenirovaniia pozvolilo legko obnaruzhit' aminokislotnuiu zamenu Y12H v odnom iz tselevykh belkov (CYB5A).

Keywords: data processing; de novo sequencing; mass spectrometry; shotgun proteomics.

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