Identification of ubiquitin/ubiquitin-like protein modification from tandem mass spectra with various PTMs
© Kang and Yi; licensee BioMed Central Ltd. 2011
Published: 14 December 2011
Various solutions have been introduced for the identification of post-translational modification (PTM) from tandem mass spectrometry (MS/MS) in proteomics field but the identification of peptide modifiers, such as Ubiquitin (Ub) and ubiquitin-like proteins (Ubls), is still a challenge. The fragmentation of peptide modifier produce complex shifted ion mass patterns in combination with other PTMs, which makes it difficult to identify and locate the PTMs on a protein sequence. Currently, most PTM identification methods do not consider the complex fragmentation of peptide modifier or deals it separately from the other PTMs.
We developed an advanced PTM identification method that inspects possible ion patterns of the most known peptide modifiers as well as other known biological and chemical PTMs to make more comprehensive and accurate conclusion. The proposed method searches all detectable mass differences of measured peaks from their theoretical values and the mass differences within mass tolerance range are grouped as mass shift classes. The most possible locations of multiple PTMs including peptide modifiers can be determined by evaluating all possible scenarios generated by the combination of the qualified mass shift classes.The proposed method showed excellent performance in the test with simulated spectra having various PTMs including peptide modifiers and in the comparison with recently developed methods such as QuickMod and SUMmOn. In the analysis of HUPO Brain Proteome Project (BPP) datasets, the proposed method could find the ubiquitin modification sites that were not identified by other conventional methods.
This work presents a novel method for identifying bothpeptide modifiers that generate complex fragmentation patternsand PTMs that are not fragmented during fragmentation processfrom tandem mass spectra.
Identification techniques of post-translational modification (PTM) from mass spectra were developed to understand the functions of PTMs in biological pathways. Ub and Ubls play important roles in regulatory networks in most of cellular processes and the importance of identification of Ub and Ubls is increased.
From tandem mass spectra, it is difficult to identify peptide with considering all possible PTMs with high performance. Standard database search algorithms (Mascot , SEQUEST [2, 3], X!Tandem ) are well-known tools for peptide identification and these database search programs can identify restricted number of modifications. PTM identification methods which can support unrestricted PTM identification are introduced to cover various PTMs. To reduce computational complexity, candidate peptides are filtered with external tools like standard database search algorithms based on peptide fragment fingerprinting (PFF) or sequence tag extraction based programs . Peptide filtering supports PTM identification tools considering various possibilities of PTMs in proper time by reducing target peptide DB or direct peptide identification. From identified peptide by SEQUEST, P-mod  calculates shifted mass from precursor ion mass and detects the sequence location of PTM. PTM-Explorer  detects PTMs with identified peptide sequence information from standard database search algorithms. Recently, QuickMod  that searches PTM using prebuilt library spectra showed favorable performance against other PTM analysis methods.
We present a new method that can identify not only peptide modifiers such as Ub and Ubls but also various PTMs from tandem mass spectra. Suggested method detects mass shift classes and identifies PTMs with considering all known peptide modifiers. All theoretical mass shifts from peptide modifiers are matched with mass shift classes from MS/MS spectra without peak filtering. The proposed method can attempt to identify peptide modifiers though there is lack of information of precursor ion mass. All known PTMs and unknown mass shifts can be considered together while peptide modifiers identification.
Unrestricted PTM identification
Identification of Ub/Ubls
Identification of mixed PTMs including Ub/Ubls
Average accuracy of the proposed method on simulated spectra datasets generated with 4 different criteria.
Biological PTMs + Ub/Ubls
Biological PTMs + Ub/Ubls
Identification of Ub/Ubls
For validation of ability of Ub/Ubls identification, mass spectra datasets from The Raught Lab Spectral Libraries that are built with SUMmOn are analyzed by the proposed method and successfully identified Ub, SUMO-1, SUMO-2/3, NEDD8, and diglycine modification from 36 mass spectra, which is exactly same with prior identification by T. Srikumar et al. . The Raught Lab Spectral Libraries contains 478 consensus spectra with 36 mono-Ub/Ubls identified mass spectra: 4 Ub, 11 NEDD8, 3 SUMO-1, 11 SUMO-2/3 and 7 diglycine modifications. Mass spectra in The Raught Lab Spectral Libraries are CID spectra with high charge states up to 7+. The proposed method designed to cover charge states up to 10+ by calculating theoretical fragment ion mass with charge state variations and cluster matched fragment ions with mass shift classes. In the Spectral Libraries, precursor ion mass is existed on each mass spectrum which can be referred for accurate PTM identification. Mass shift paths that satisfy precursor ion mass are selected and PTMs in mass shift paths are identified by the proposed method. While T. Srikumar et al. used two PTM identification programs, X!Tandem for identification of diglycine modifications and SUMmOn for identification of Ub, NEDD8, SUMO-1, and SUMO-2/3, the proposed method successfully identified diglycine and Ub/Ubls without any other PTM identification program.
Identification of PTMs
For validation of ability of PTM identification, mass spectra datasets that are used for QuickMod are analyzed by the proposed method. QuickMod searches PTMs using prebuilt library spectra, while the proposed method is based on theoretical peptide spectra match. Prebuilt library spectra can boost up identification accuracy but hard to cover mass spectra that are not existed in spectrum library. QuickMod provided test data sets that cover 5 PTMs (oxidation of methionine, phosphorylation of serine, threonine, or tyrosine, carbamidomethylation of cysteine, n-term acetylation and pyro-glu on n-term glutamic acid or glutamine) by extracting spectra from spectral libraries publicly available at NIST (http://peptide.nist.gov/) and ISB (http://www.peptideatlas.org). Only one PTM is existed per spectrum in test data sets as QuickMod is not designed for identifying multiple PTMs. The proposed method analyzed doubly charged spectra (Mod_z2) from QuickMod test data sets with unrestricted search criteria. The accuracy of QuickMod with position agreement policy using theoretical peptide spectra match was 94%, while the accuracy of the proposed method with unrestricted search by theoretical peptide spectra match was 92.48%. The proposed method showed comparable performance of PTM identification with QuickMod, recently introduced PTM identification method.
Detection of ubiquitination from tandem mass spectra datasets
Identification of peptide modifiers such as ubiquitin and ubiquitin-like proteins requires additional techniques to handle various mass shifts made by fragments of peptide modifiers. The proposed method showed the ability of identification of multiple PTMs including peptide modifiers. The proposed method can be applied on analyzing tandem mass spectra datasets built with standard database search algorithms. Though precursor ion mass can help accurate identification of PTMs, the proposed method can try to identify PTMs when there are lack of information of precursor ion mass in the mass spectra. Applied to cluster computing, our method can analyze massive mass spectra datasets from high-throughput experiments. With development of mass spectrometer in terms of accuracy and sensitivity, overall performance of our method will be dramatically increased because smaller tolerance can effectively reduce computational artifacts of mass shift classes.
Though identification of peptide modifiers becomes important to understand their roles in biological pathway regulations, identification of peptide modifiers with complex peak patterns from fragment ions of peptide modifiers remains a challenge. In this paper, we introduce a method for identification of peptide modifiers from tandem mass spectra with various PTMs with considering possible clues based on unrestricted PTM identification algorithm previously developed by authors and fragmented ion patterns of peptide modifiers. The proposed method is a novel method that can identify both PTMs that are not fragmented during fragmentation process and peptide modifiers that generate complex fragmentation patterns together from tandem mass spectra. Proposed method showed excellent performance in the test with simulated spectra having various PTMs including peptide modifiers and in the comparison with methods specialized for identification of PTMs or peptide modifiers. Not only identification of mono-Ub/Ubl or mono-PTM, identification of multiple PTMs including all known peptide modifiers can be applied with the proposed method.
This work was partially supported by the Converging Research Center Program funded by the Ministry of Education, Science and Technology of Korea (Project No. 2011K000864), by the National Research Foundation (NRF) grant (No. 2011-0018264) of Ministry of Education, Science and Technology of Korea, and by the Korea Institute of Science and Technology Information (KISTI).
This article has been published as part of BMC Bioinformatics Volume 12 Supplement 14, 2011: 22nd International Conference on Genome Informatics: Bioinformatics. The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2105/12?issue=S14.
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