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Improved identification of O-linked glycopeptides from ETD data with optimized scoring for different charge states and cleavage specificities.

Darula Z, Chalkley RJ, Lynn A, Baker PR, Medzihradszky KF - Amino Acids (2010)

Bottom Line: Interpretation of the corresponding ETD data using Protein Prospector is also presented.We show that the improved scoring is more than doubled the glycopeptide assignments under very strict acceptance criteria.This study illustrates that "old" proteomic data may yield significant new information when re-interrogated with new, improved tools.

View Article: PubMed Central - PubMed

Affiliation: Proteomics Research Group, Biological Research Center, 62 Temesvari krt, Szeged, 6726, Hungary.

ABSTRACT
This article describes the effect of re-interrogation of electron-transfer dissociation (ETD) data with newly developed analytical tools. MS/MS-based characterization of O-linked glycopeptides is discussed using data acquired from a complex mixture of O-linked glycopeptides, featuring mucin core 1-type carbohydrates with and without sialic acid, as well as after partial deglycosylation to leave only the core GalNAc units (Darula and Medzihradszky in Mol Cell Proteomics 8:2515, 2009). Information content of collision-induced dissociation spectra generated in collision cell (in QqTOF instruments) and in ion traps is compared. Interpretation of the corresponding ETD data using Protein Prospector is also presented. Search results using scoring based on the frequency of different fragment ions occurring in ETD spectra of tryptic peptides are compared with results obtained after ion weightings were adjusted to accommodate differential ion frequencies in spectra of differing charge states or cleavage specificities. We show that the improved scoring is more than doubled the glycopeptide assignments under very strict acceptance criteria. This study illustrates that "old" proteomic data may yield significant new information when re-interrogated with new, improved tools.

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Linear ion trap ETD spectrum of m/z 806.3813 (3+) acquired during the analysis of the partially deglycosylated mixture. The corresponding structure was identified as 159IALPT(GalNAc)QDPATHGGAS(GalNAc)SKASSD179 of insulin-like growth factor 2. Asterisks indicate c − 1• and z + 1 ions. The ion at m/z 1610 is the charge-reduced ion for a 2+ ion within the precursor selection window
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Fig3: Linear ion trap ETD spectrum of m/z 806.3813 (3+) acquired during the analysis of the partially deglycosylated mixture. The corresponding structure was identified as 159IALPT(GalNAc)QDPATHGGAS(GalNAc)SKASSD179 of insulin-like growth factor 2. Asterisks indicate c − 1• and z + 1 ions. The ion at m/z 1610 is the charge-reduced ion for a 2+ ion within the precursor selection window

Mentions: New glycosylation sites identified in this study


Improved identification of O-linked glycopeptides from ETD data with optimized scoring for different charge states and cleavage specificities.

Darula Z, Chalkley RJ, Lynn A, Baker PR, Medzihradszky KF - Amino Acids (2010)

Linear ion trap ETD spectrum of m/z 806.3813 (3+) acquired during the analysis of the partially deglycosylated mixture. The corresponding structure was identified as 159IALPT(GalNAc)QDPATHGGAS(GalNAc)SKASSD179 of insulin-like growth factor 2. Asterisks indicate c − 1• and z + 1 ions. The ion at m/z 1610 is the charge-reduced ion for a 2+ ion within the precursor selection window
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102200&req=5

Fig3: Linear ion trap ETD spectrum of m/z 806.3813 (3+) acquired during the analysis of the partially deglycosylated mixture. The corresponding structure was identified as 159IALPT(GalNAc)QDPATHGGAS(GalNAc)SKASSD179 of insulin-like growth factor 2. Asterisks indicate c − 1• and z + 1 ions. The ion at m/z 1610 is the charge-reduced ion for a 2+ ion within the precursor selection window
Mentions: New glycosylation sites identified in this study

Bottom Line: Interpretation of the corresponding ETD data using Protein Prospector is also presented.We show that the improved scoring is more than doubled the glycopeptide assignments under very strict acceptance criteria.This study illustrates that "old" proteomic data may yield significant new information when re-interrogated with new, improved tools.

View Article: PubMed Central - PubMed

Affiliation: Proteomics Research Group, Biological Research Center, 62 Temesvari krt, Szeged, 6726, Hungary.

ABSTRACT
This article describes the effect of re-interrogation of electron-transfer dissociation (ETD) data with newly developed analytical tools. MS/MS-based characterization of O-linked glycopeptides is discussed using data acquired from a complex mixture of O-linked glycopeptides, featuring mucin core 1-type carbohydrates with and without sialic acid, as well as after partial deglycosylation to leave only the core GalNAc units (Darula and Medzihradszky in Mol Cell Proteomics 8:2515, 2009). Information content of collision-induced dissociation spectra generated in collision cell (in QqTOF instruments) and in ion traps is compared. Interpretation of the corresponding ETD data using Protein Prospector is also presented. Search results using scoring based on the frequency of different fragment ions occurring in ETD spectra of tryptic peptides are compared with results obtained after ion weightings were adjusted to accommodate differential ion frequencies in spectra of differing charge states or cleavage specificities. We show that the improved scoring is more than doubled the glycopeptide assignments under very strict acceptance criteria. This study illustrates that "old" proteomic data may yield significant new information when re-interrogated with new, improved tools.

Show MeSH
Related in: MedlinePlus