Introduction: Oleosins are proteins associated to oil bodies of seeds and investigated because demonstrated to be responsible of severe allergic reaction in allergic patients. During the characterization of hazelnut oleosins, selected by immunoreaction with the sera of allergic Italian children, the analyses with two MS instruments improved our capacity of data interpretation and allowed to identified a new isoform probably more reactive than the two oleosins already known. Methods: A protocol for the extraction of oil bodies associated proteins was optimized and SDS-PAGE was performed. The PMF method ("in gel" trypsin digestion, peptide collection and MS analysis) was applied to the bands at 14 and 17 kDa. All protein bands were identified by both MALDI-TOF/TOF and LC-MS/MS analyses. MALDI-TOF/TOF spectra were manually interpreted, if necessary, and results were used to refine LC-MS/MS interpretation. Results: The electrophoresis profile of the oil bodies associated proteins showed two most abundant bands. MALDI TOF/TOF analysis was performed on bands at 14 and 17 kDa from SDS-PAGE of hazelnut extract, where 100% of protein content resulted to be oleosins Cora12 and 13, already known as allergens. The LC-MS/MS analysis confirmed the same results. However, the manual checking of major signals of the MALDI-TOF spectra of the band at 17kDa revealed that only minor signals identified the oleosin Cor a 12 and that at least two high intensity signals were without any protein attribution. The fragmentation spectra from LIFT-TOF/TOF of two major picks were analyzed using MS-Tag Protein Prospector software in identity mode and didn't give any protein identification. When the software was set in homology mode with Cor a 12 as template, a new N-terminal was revealed indicating the presence a new isoform not present in the NCBI database. Conclusions: In protein identification the dependence of MS techniques from the DNA and protein database is very strong and new isoforms are rarely identified. In this work a new isoform of oleosins from hazelnut was identified combining manual and software-assisted interpretation of MALDI LIFT-TOF/TOF spectra. Novel Aspect: Sometimes old practices like to have look at spectra and try to assign almost all signals by manual interpretation of MS/MS spectra allow to really discover something new that normally miss out on automatic data interpretation.
The synergism of MALDI LIFT-TOF/TOF MS and ESI -Q-TOF MS instruments to discover new isoforms of allergens from hazelnut oleosin extract.
Nebbia S;Lamberti C;
2018
Abstract
Introduction: Oleosins are proteins associated to oil bodies of seeds and investigated because demonstrated to be responsible of severe allergic reaction in allergic patients. During the characterization of hazelnut oleosins, selected by immunoreaction with the sera of allergic Italian children, the analyses with two MS instruments improved our capacity of data interpretation and allowed to identified a new isoform probably more reactive than the two oleosins already known. Methods: A protocol for the extraction of oil bodies associated proteins was optimized and SDS-PAGE was performed. The PMF method ("in gel" trypsin digestion, peptide collection and MS analysis) was applied to the bands at 14 and 17 kDa. All protein bands were identified by both MALDI-TOF/TOF and LC-MS/MS analyses. MALDI-TOF/TOF spectra were manually interpreted, if necessary, and results were used to refine LC-MS/MS interpretation. Results: The electrophoresis profile of the oil bodies associated proteins showed two most abundant bands. MALDI TOF/TOF analysis was performed on bands at 14 and 17 kDa from SDS-PAGE of hazelnut extract, where 100% of protein content resulted to be oleosins Cora12 and 13, already known as allergens. The LC-MS/MS analysis confirmed the same results. However, the manual checking of major signals of the MALDI-TOF spectra of the band at 17kDa revealed that only minor signals identified the oleosin Cor a 12 and that at least two high intensity signals were without any protein attribution. The fragmentation spectra from LIFT-TOF/TOF of two major picks were analyzed using MS-Tag Protein Prospector software in identity mode and didn't give any protein identification. When the software was set in homology mode with Cor a 12 as template, a new N-terminal was revealed indicating the presence a new isoform not present in the NCBI database. Conclusions: In protein identification the dependence of MS techniques from the DNA and protein database is very strong and new isoforms are rarely identified. In this work a new isoform of oleosins from hazelnut was identified combining manual and software-assisted interpretation of MALDI LIFT-TOF/TOF spectra. Novel Aspect: Sometimes old practices like to have look at spectra and try to assign almost all signals by manual interpretation of MS/MS spectra allow to really discover something new that normally miss out on automatic data interpretation.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
