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. 2017 Jul;63(4):341-350.
doi: 10.18097/PBMC20176304341.

[Use of de novo sequencing for proteins identification]

[Article in Russian]
V S Skvortsov  1 , A V Mikurova  1 , A V Rybina  1
Affiliations

[Use of de novo sequencing for proteins identification]

[Article in Russian]
V S Skvortsov et al. Biomed Khim. 2017 Jul.

Abstract
in English, Russian

Three de novo sequencing programs (Novor, PEAKS and PepNovo+) have been used for identification of 48 individual human proteins constituting the Universal Proteomics Standard Set 2 (UPS2) ("Sigma-Aldrich", USA). Experimental data have been obtained by tandem mass spectrometry. The MS/MS was performed using pure UPS2 and UPS2 mixtures with E. coli extract and human plasma samples. Protein detection was based on identification of at least two peptides of 9 residues in length or one peptide containing at least 13 residues. Using these criteria 13 (Novor), 20 (PEAKS) and 11 (PepNovo+) proteins were detected in pure UPS2 sample. Protein identifications in mixed samples were comparable or worse. Better results (by ~20%) were obtained using prediction included high quality identified fragment (TAG) containing at least 7 residues and unidentified additional masses at N- and C-termini (PepNovo+). The latter approach confidently recognized mass-spectrometric artefacts (and probably PTM). Atypical mass changes missed in UNIMOD DB were found (PepNovo+) to be statistically significant at the C-terminus (+23.02, +26.04 and +27.03). Using peptides containing these modifications and milder detection threshold 41 of 48 UPS2 proteins were identified.

S ispol'zovaniem trekh programm de novo sekvenirovaniia (Novor, PEAKS i PepNovo+) provedena identifikatsiia belkov iz nabora, vkliuchaiushchego 48 belkov cheloveka (nabor UPS2 kompanii “Sigma-Aldrich”, SShA). Éksperimental'nye dannye polucheny pri pomoshchi tandemnoĭ mass-spektrometrii. V rabote issledovany kak nabor dannykh belkov, tak i kombinirovannye proby s dobavleniem ékstrakta E. coli i plazmy krovi cheloveka. Pri ispol'zovanii kriteriia identifikatsii (dva peptida dlinoĭ ne menee deviati ostatkov ili odin peptid ne koroche 13 ostatkov) v “chistoĭ” probe obnaruzheno 13 (Novor), 20 (Peaks) i 11 (PepNovo+) belkov iz nabora UPS2. Dlia kombinirovannykh prob rezul'tat byl ili sopostavimym, ili khuzhe. Pri ispol'zovanii predskazaniĭ, vkliuchaiushchikh vysokodostovernyĭ fragment (TAG) posledovatel'nosti dlinoĭ ne menee 7 ostatkov, i “ostatochnye” (neidentifitsirovannye) massy s N- i S-kontsov (PepNovo+), rezul'tat sushchestvenno uluchshaetsia (~20%) i khorosho identifitsiruiutsia mass-spektrometricheskie artefakty i, veroiatno, PTM. V rabote zaregistrirovany nestandartnye S-kontsevye izmeneniia mass (+23,02, +26,04 i +27,03) vstrechaiushchiesia statisticheski dostoverno. Pri ispol'zovanii peptidov, soderzhashchikh éti modifikatsii, a takzhe pogranichnykh kriteriev identifikatsii, v razlichnykh probakh udalos' obnaruzhit' 41 iz 48 belkov nabora UPS2.

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

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