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Improved protein identification efficiency by mass spectrometry using N-terminal chemical derivatization of peptides from Angiostrongylus costaricensis, a nematode with unknown genome.
J Mass Spectrom. 2007 Oct; 42(10):1363-74.JM

Abstract

Matrix-assisted laser desorption ionization (MALDI), Peptide Mass Fingerprinting (PMF) and MALDI-MS/MS ion search (using MASCOT) have become the preferred methods for high-throughput identification of proteins. Unfortunately, PMF can be ambiguous, mainly when the genome of the organism under investigation is unknown and the quality of spectra generated is poor and does not allow confident identification. The post-source decay (PSD) fragmentation of singly charged tryptic peptide ions generated by MALDI-TOF/TOF typically results in low fragmentation efficiency and/or complex spectra, including backbone fragmentation ions (series b and y), internal fragmentation etc. Interpreting these data either manually and/or using de novo sequencing software can frequently be a challenge. To overcome this limitation when studying the proteome of adult Angiostrongylus costaricensis, a nematode with unknown genome, we have used chemical N-terminal derivatization of the tryptic peptides with 4-sulfophenyl isothiocyanate (SPITC) prior to MALDI-TOF/TOF MS. This methodology has recently been reported to enhance the quality of MALDI-TOF/TOF-PSD data, allowing the obtainment of complete sequence of most of the peptides and thus facilitating de novo peptide sequencing. Our approach, consisting of SPITC derivatization along with manual spectra interpretation and Blast analysis, was able to positively identify 76% of analyzed samples, whereas MASCOT analysis of derivatized samples, MASCOT analysis of nonderivatized samples and PMF of nonderivatized samples yielded only 35, 41 and 12% positive identifications, respectively. Moreover, de novo sequencing of SPITC modified peptides resulted in protein sequences not available in NCBInr database paving the way to the discovery of new protein molecules.

Authors+Show Affiliations

Department of Physiology and Pharmacodynamics, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

17902111

Citation

León, Ileana R., et al. "Improved Protein Identification Efficiency By Mass Spectrometry Using N-terminal Chemical Derivatization of Peptides From Angiostrongylus Costaricensis, a Nematode With Unknown Genome." Journal of Mass Spectrometry : JMS, vol. 42, no. 10, 2007, pp. 1363-74.
León IR, Neves-Ferreira AG, Valente RH, et al. Improved protein identification efficiency by mass spectrometry using N-terminal chemical derivatization of peptides from Angiostrongylus costaricensis, a nematode with unknown genome. J Mass Spectrom. 2007;42(10):1363-74.
León, I. R., Neves-Ferreira, A. G., Valente, R. H., Mota, E. M., Lenzi, H. L., & Perales, J. (2007). Improved protein identification efficiency by mass spectrometry using N-terminal chemical derivatization of peptides from Angiostrongylus costaricensis, a nematode with unknown genome. Journal of Mass Spectrometry : JMS, 42(10), 1363-74.
León IR, et al. Improved Protein Identification Efficiency By Mass Spectrometry Using N-terminal Chemical Derivatization of Peptides From Angiostrongylus Costaricensis, a Nematode With Unknown Genome. J Mass Spectrom. 2007;42(10):1363-74. PubMed PMID: 17902111.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Improved protein identification efficiency by mass spectrometry using N-terminal chemical derivatization of peptides from Angiostrongylus costaricensis, a nematode with unknown genome. AU - León,Ileana R, AU - Neves-Ferreira,Ana G C, AU - Valente,Richard H, AU - Mota,Ester M, AU - Lenzi,Henrique L, AU - Perales,Jonas, PY - 2007/9/29/pubmed PY - 2007/12/7/medline PY - 2007/9/29/entrez SP - 1363 EP - 74 JF - Journal of mass spectrometry : JMS JO - J Mass Spectrom VL - 42 IS - 10 N2 - Matrix-assisted laser desorption ionization (MALDI), Peptide Mass Fingerprinting (PMF) and MALDI-MS/MS ion search (using MASCOT) have become the preferred methods for high-throughput identification of proteins. Unfortunately, PMF can be ambiguous, mainly when the genome of the organism under investigation is unknown and the quality of spectra generated is poor and does not allow confident identification. The post-source decay (PSD) fragmentation of singly charged tryptic peptide ions generated by MALDI-TOF/TOF typically results in low fragmentation efficiency and/or complex spectra, including backbone fragmentation ions (series b and y), internal fragmentation etc. Interpreting these data either manually and/or using de novo sequencing software can frequently be a challenge. To overcome this limitation when studying the proteome of adult Angiostrongylus costaricensis, a nematode with unknown genome, we have used chemical N-terminal derivatization of the tryptic peptides with 4-sulfophenyl isothiocyanate (SPITC) prior to MALDI-TOF/TOF MS. This methodology has recently been reported to enhance the quality of MALDI-TOF/TOF-PSD data, allowing the obtainment of complete sequence of most of the peptides and thus facilitating de novo peptide sequencing. Our approach, consisting of SPITC derivatization along with manual spectra interpretation and Blast analysis, was able to positively identify 76% of analyzed samples, whereas MASCOT analysis of derivatized samples, MASCOT analysis of nonderivatized samples and PMF of nonderivatized samples yielded only 35, 41 and 12% positive identifications, respectively. Moreover, de novo sequencing of SPITC modified peptides resulted in protein sequences not available in NCBInr database paving the way to the discovery of new protein molecules. SN - 1076-5174 UR - https://www.unboundmedicine.com/medline/citation/17902111/Improved_protein_identification_efficiency_by_mass_spectrometry_using_N_terminal_chemical_derivatization_of_peptides_from_Angiostrongylus_costaricensis_a_nematode_with_unknown_genome_ DB - PRIME DP - Unbound Medicine ER -