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De novo sequencing using MELD proteolysis coupled to a "sequence assembly" algorithm
Mazzucchelli, Gabriel; Zimmerman, Tyler; Smargiasso, Nicolas et al.
2015In 63rd ASMS Conference Proceedings, May 30 - June 4 2015, St. Louis, MO
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Keywords :
de novo sequencing; Mass spectrometry; protein digestion; multi enzymatic digestion; MELD; multi enzymatic limited digestion; sequence assembly; TAILS
Abstract :
[en] Introduction Protein de novo sequencing requires a method that combines extensive MSMS fragmentation and an appropriate data processing. This can be applied either on intact protein or on its proteolytic peptides. Peptide analysis has the advantage to be compatible with well-known workflows. Nevertheless the connectivity between peptides is lost. Taking these considerations into account, a specific digestion method and a sequence assembly software were developed. The MELD method relies on a combination of MultiEnzymatic AND Limited proteolytic Digestions. The MELD generates in a single experiment numerous different peptides with miss-cleavages that overlap when aligned on the matching protein sequence. The two major benefices are an increased probability to obtain the entire protein sequence and a redundancy in the protein sequence matches. Methods The MELD consists in two parallel 2h digestions both using an optimized protease mixture. The mixtures are composed of the same proteases but in different relative quantities. Analyses were performed by UPLC-Orbitrap (IClass, Waters, QExactive, Thermo). Data were processed with PEAKS (BSI) to generate de novo sequence candidates. After data importation into our software, a seed sequence is set or can be found automatically. The software extends the seed sequence in both directions with moving windows of three amino acids, plus a fourth one to be added. The added amino acid is validated in several ways, including by the total frequency of occurrence, by larger windows of aa, by the local spectrum-derived confidence, and by combinations of these factors. Preliminary Data The MELD protocol was first validated by applying a traditional database search workflow on several commercial proteins with an inter-day and inter-individual procedure. These experiments showed 100% sequence coverage for each protein analyzed, involving information on peptide identity and modifications localization. Strong confident identification was obtained due to multiple overlapping peptides matches with the given sequence and with a high number of overlapping peptides assignments. The analysis of the four proteins provided the following results: HSA, Myoglobin and Lysozyme were identified with 100% sequence coverage with respectively 890, 300 and 120 unique peptides (CV<10%, peptide FDR<0.1%). The variable region of each heavy and light chains of Adalimumab antibody were identified with 100% sequence coverage. With the MELD, an average of 10 different peptides covering each sequence stretch of the protein could be obtained. The combinatory effect of the multiple enzymes used and the limited digestions leads to an increased robustness, very high confidence identifications and allows clear localization of PTMs. The MELD protocol as presented here and tested on several pure proteins digested in solution, certainly improves the general "bottom-up" strategy applied for highly confident protein identification and would allow better protein characterization, even for those having PTMs. In addition, in our analysis, each fragment position of the entire protein sequence was evidenced either by a "y" or a "b" fragment ion. This high and confident amount of information enables extensive de novo sequencing using PEAKS software, followed by application of our “sequence assembly” algorithm. The first version of our assembling tool on MELD experimental data generated long sequence tags, up to 90 amino acids long.
Disciplines :
Chemistry
Author, co-author :
Mazzucchelli, Gabriel  ;  Université de Liège > Département de chimie (sciences) > Laboratoire de spectrométrie de masse (L.S.M.)
Zimmerman, Tyler;  National Institute of Standards and Technology - Gaithersburg, MD, USA > Mass Spectrometry Data Center Group
Smargiasso, Nicolas ;  Université de Liège > Département de chimie (sciences) > Laboratoire de spectrométrie de masse (L.S.M.)
Baiwir, Dominique  ;  Université de Liège > GIGA-Management : Plate-forme protéomique
MEUWIS, Marie-Alice  ;  Centre Hospitalier Universitaire de Liège - CHU > Gastro-Entérologie-Hépatologie
De Pauw, Edwin  ;  Université de Liège > Département de chimie (sciences) > Laboratoire de spectrométrie de masse (L.S.M.)
Language :
English
Title :
De novo sequencing using MELD proteolysis coupled to a "sequence assembly" algorithm
Publication date :
June 2015
Event name :
63rd ASMS Conference on Mass Spectrometry & Allied Topics
Event place :
St Louis, MO, United States
Event date :
du 30 mai au 04 Juin 2015
Audience :
International
Main work title :
63rd ASMS Conference Proceedings, May 30 - June 4 2015, St. Louis, MO
Peer reviewed :
Peer reviewed
Available on ORBi :
since 15 June 2015

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