Correction of errors in tandem mass spectrum extraction enhances phosphopeptide identification

The tandem mass spectrum extraction of phosphopeptides is more difficult and error-prone than that of unmodified peptides due to their lower abundance, lower ionization efficiency, the cofragmentation with other high-abundance peptides, and the use of MS3 on MS2 fragments with neutral losses. Howeve...

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Main Authors: Hao, Piliang, Ren, Yan, Sze, Siu Kwan, Tam, James P.
Other Authors: School of Biological Sciences
Format: Article
Language:English
Published: 2014
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Online Access:https://hdl.handle.net/10356/103597
http://hdl.handle.net/10220/19341
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Institution: Nanyang Technological University
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spelling sg-ntu-dr.10356-1035972023-02-28T17:05:50Z Correction of errors in tandem mass spectrum extraction enhances phosphopeptide identification Hao, Piliang Ren, Yan Sze, Siu Kwan Tam, James P. School of Biological Sciences Singapore Centre for Environmental Life Sciences Engineering DRNTU::Science::Biological sciences The tandem mass spectrum extraction of phosphopeptides is more difficult and error-prone than that of unmodified peptides due to their lower abundance, lower ionization efficiency, the cofragmentation with other high-abundance peptides, and the use of MS3 on MS2 fragments with neutral losses. However, there are still no established methods to evaluate its correctness. Here we propose to identify and correct these errors via the combinatorial use of multiple spectrum extraction tools. We evaluated five free and two commercial extraction tools using Mascot and phosphoproteomics raw data from LTQ FT Ultra, in which RawXtract 1.9.9.2 identified the highest number of unique phosphopeptides (peptide expectation value <0.05). Surprisingly, ProteoWizzard (v. 3.0.3476) extracted wrong precursor mass for most MS3 spectra. Comparison of the top three free extraction tools showed that only 54% of the identified spectra were identified consistently from all three tools, indicating that some errors might happen during spectrum extraction. Manual check of 258 spectra not identified from all three tools revealed 405 errors of spectrum extraction with 7.4% in selecting wrong precursor charge, 50.6% in selecting wrong precursor mass, and 42.1% in exporting MS/MS fragments. We then corrected the errors by selecting the best extracted MGF file for each spectrum among the three tools for another database search. With the errors corrected, it results in the 22.4 and 12.2% increase in spectrum matches and unique peptide identification, respectively, compared with the best single method. Correction of errors in spectrum extraction improves both the sensitivity and confidence of phosphopeptide identification. Data analysis on nonphosphopeptide spectra indicates that this strategy applies to unmodified peptides as well. The identification of errors in spectrum extraction will promote the improvement of spectrum extraction tools in future. NMRC (Natl Medical Research Council, S’pore) Accepted version 2014-05-15T05:53:23Z 2019-12-06T21:16:06Z 2014-05-15T05:53:23Z 2019-12-06T21:16:06Z 2013 2013 Journal Article Hao, P., Ren, Y., Tam, J. P., & Sze, S. K. (2013). Correction of Errors in Tandem Mass Spectrum Extraction Enhances Phosphopeptide Identification. Journal of Proteome Research, 12(12), 5548-5557. 1535-3893 https://hdl.handle.net/10356/103597 http://hdl.handle.net/10220/19341 10.1021/pr4004486 178794 en Journal of proteome research © 2013 American Chemical Society. This is the author created version of a work that has been peer reviewed and accepted for publication by Journal of Proteome Research, American Chemical Society. It incorporates referee’s comments but changes resulting from the publishing process, such as copyediting, structural formatting, may not be reflected in this document. The published version is available at: [http://dx.doi.org/10.1021/pr4004486]. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic DRNTU::Science::Biological sciences
spellingShingle DRNTU::Science::Biological sciences
Hao, Piliang
Ren, Yan
Sze, Siu Kwan
Tam, James P.
Correction of errors in tandem mass spectrum extraction enhances phosphopeptide identification
description The tandem mass spectrum extraction of phosphopeptides is more difficult and error-prone than that of unmodified peptides due to their lower abundance, lower ionization efficiency, the cofragmentation with other high-abundance peptides, and the use of MS3 on MS2 fragments with neutral losses. However, there are still no established methods to evaluate its correctness. Here we propose to identify and correct these errors via the combinatorial use of multiple spectrum extraction tools. We evaluated five free and two commercial extraction tools using Mascot and phosphoproteomics raw data from LTQ FT Ultra, in which RawXtract 1.9.9.2 identified the highest number of unique phosphopeptides (peptide expectation value <0.05). Surprisingly, ProteoWizzard (v. 3.0.3476) extracted wrong precursor mass for most MS3 spectra. Comparison of the top three free extraction tools showed that only 54% of the identified spectra were identified consistently from all three tools, indicating that some errors might happen during spectrum extraction. Manual check of 258 spectra not identified from all three tools revealed 405 errors of spectrum extraction with 7.4% in selecting wrong precursor charge, 50.6% in selecting wrong precursor mass, and 42.1% in exporting MS/MS fragments. We then corrected the errors by selecting the best extracted MGF file for each spectrum among the three tools for another database search. With the errors corrected, it results in the 22.4 and 12.2% increase in spectrum matches and unique peptide identification, respectively, compared with the best single method. Correction of errors in spectrum extraction improves both the sensitivity and confidence of phosphopeptide identification. Data analysis on nonphosphopeptide spectra indicates that this strategy applies to unmodified peptides as well. The identification of errors in spectrum extraction will promote the improvement of spectrum extraction tools in future.
author2 School of Biological Sciences
author_facet School of Biological Sciences
Hao, Piliang
Ren, Yan
Sze, Siu Kwan
Tam, James P.
format Article
author Hao, Piliang
Ren, Yan
Sze, Siu Kwan
Tam, James P.
author_sort Hao, Piliang
title Correction of errors in tandem mass spectrum extraction enhances phosphopeptide identification
title_short Correction of errors in tandem mass spectrum extraction enhances phosphopeptide identification
title_full Correction of errors in tandem mass spectrum extraction enhances phosphopeptide identification
title_fullStr Correction of errors in tandem mass spectrum extraction enhances phosphopeptide identification
title_full_unstemmed Correction of errors in tandem mass spectrum extraction enhances phosphopeptide identification
title_sort correction of errors in tandem mass spectrum extraction enhances phosphopeptide identification
publishDate 2014
url https://hdl.handle.net/10356/103597
http://hdl.handle.net/10220/19341
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