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Increasing the productivity of glycopeptides analysis by using higher-energy collision dissociation-accurate mass-product-dependent electron transfer dissociation.

Saba J, Dutta S, Hemenway E, Viner R - Int J Proteomics (2012)

Bottom Line: ETD spectra are only acquired when glycan oxonium ions from MS/MS HCD are detected.The advantage of this approach is that it streamlines data analysis and improves dynamic range and duty cycle.Here, we present the benefits of HCD-PD-ETD relative to the traditional alternating HCD/ETD for a trainer set containing twelve-protein mixture with two glycoproteins: human serotransferrin, ovalbumin and contaminations of two other: bovine alpha 1 acid glycoprotein (bAGP) and bovine fetuin.

View Article: PubMed Central - PubMed

Affiliation: Thermo Fisher Scientific, San Jose, CA 95134, USA.

ABSTRACT
Currently, glycans are attracting attention from the scientific community as potential biomarkers or as posttranslational modifications (PTMs) of therapeutic proteins. However, structural characterization of glycoproteins and glycopeptides remains analytically challenging. Here, we report on the implementation of a novel acquisition strategy termed higher-energy collision dissociation-accurate mass-product-dependent electron transfer dissociation (HCD-PD-ETD) on a hybrid linear ion trap-orbitrap mass spectrometer. This acquisition strategy uses the complementary fragmentations of ETD and HCD for glycopeptides analysis in an intelligent fashion. Furthermore, the approach minimizes user input for optimizing instrumental parameters and enables straightforward detection of glycopeptides. ETD spectra are only acquired when glycan oxonium ions from MS/MS HCD are detected. The advantage of this approach is that it streamlines data analysis and improves dynamic range and duty cycle. Here, we present the benefits of HCD-PD-ETD relative to the traditional alternating HCD/ETD for a trainer set containing twelve-protein mixture with two glycoproteins: human serotransferrin, ovalbumin and contaminations of two other: bovine alpha 1 acid glycoprotein (bAGP) and bovine fetuin.

No MeSH data available.


Related in: MedlinePlus

LC-MS ion trap ETD spectrum of bovine fetuin N-linked glycopeptide T72–103 precursor at m/z 1219.950 (5+).
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Related In: Results  -  Collection


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fig4: LC-MS ion trap ETD spectrum of bovine fetuin N-linked glycopeptide T72–103 precursor at m/z 1219.950 (5+).

Mentions: To demonstrate the advantage of our novel acquisition strategy, we selected a twelve-protein mixture containing two glycoproteins: human serotransferrin and ovalbumin and contaminants of two others: bAGP and bovine fetuin. The mixture was digested with trypsin and analyzed by the traditional alternating HCD/ETD approach and the novel HCD-PD-ETD approach. Enrichment steps were avoided prior to analysis to ensure that the glycopeptides would be very low in abundances; this gave us the opportunity to evaluate the dynamic range of analysis for both acquisition strategies. Of the two, only HCD-PD-ETD approach identified glycopeptides from the contaminant glycoproteins (bAGP and bovine fetuin) present in the sample (Figure 2). While the HCD/ETD acquisition strategy identified numerous peptides and expected glycopeptides (eluted at 34, 40, and 48 min as shown in Figure 2 for HCD-PD-ETD approach) from serotransferrin and ovalbumin, it was unable to identify glycopeptides from the contaminant glycoproteins. Manual inspection of the RAW data confirmed this, as precursors associated with the contaminant glycopeptides were not targeted for MS/MS by HCD or ETD in the alternating approach. Analysis of the HCD-PD-ETD data revealed the identification of glycopeptides from expected glycoproteins and the contaminant glycoproteins. Identified minor glycopeptides from the contaminant glycoproteins by HCD-PD-ETD acquisition method are presented in Table 1, and representative ETD MS/MS spectra used in identification are shown in Figures 3 and 4. Because HCD-PD-ETD acquisition strategy did not waste time acquiring ETD data for nonglycosylated peptides, it was able to dig deeper into the sample and identify this low abundant glycopeptides that the alternating HCD/ETD acquisition strategy could not target.


Increasing the productivity of glycopeptides analysis by using higher-energy collision dissociation-accurate mass-product-dependent electron transfer dissociation.

Saba J, Dutta S, Hemenway E, Viner R - Int J Proteomics (2012)

LC-MS ion trap ETD spectrum of bovine fetuin N-linked glycopeptide T72–103 precursor at m/z 1219.950 (5+).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369405&req=5

fig4: LC-MS ion trap ETD spectrum of bovine fetuin N-linked glycopeptide T72–103 precursor at m/z 1219.950 (5+).
Mentions: To demonstrate the advantage of our novel acquisition strategy, we selected a twelve-protein mixture containing two glycoproteins: human serotransferrin and ovalbumin and contaminants of two others: bAGP and bovine fetuin. The mixture was digested with trypsin and analyzed by the traditional alternating HCD/ETD approach and the novel HCD-PD-ETD approach. Enrichment steps were avoided prior to analysis to ensure that the glycopeptides would be very low in abundances; this gave us the opportunity to evaluate the dynamic range of analysis for both acquisition strategies. Of the two, only HCD-PD-ETD approach identified glycopeptides from the contaminant glycoproteins (bAGP and bovine fetuin) present in the sample (Figure 2). While the HCD/ETD acquisition strategy identified numerous peptides and expected glycopeptides (eluted at 34, 40, and 48 min as shown in Figure 2 for HCD-PD-ETD approach) from serotransferrin and ovalbumin, it was unable to identify glycopeptides from the contaminant glycoproteins. Manual inspection of the RAW data confirmed this, as precursors associated with the contaminant glycopeptides were not targeted for MS/MS by HCD or ETD in the alternating approach. Analysis of the HCD-PD-ETD data revealed the identification of glycopeptides from expected glycoproteins and the contaminant glycoproteins. Identified minor glycopeptides from the contaminant glycoproteins by HCD-PD-ETD acquisition method are presented in Table 1, and representative ETD MS/MS spectra used in identification are shown in Figures 3 and 4. Because HCD-PD-ETD acquisition strategy did not waste time acquiring ETD data for nonglycosylated peptides, it was able to dig deeper into the sample and identify this low abundant glycopeptides that the alternating HCD/ETD acquisition strategy could not target.

Bottom Line: ETD spectra are only acquired when glycan oxonium ions from MS/MS HCD are detected.The advantage of this approach is that it streamlines data analysis and improves dynamic range and duty cycle.Here, we present the benefits of HCD-PD-ETD relative to the traditional alternating HCD/ETD for a trainer set containing twelve-protein mixture with two glycoproteins: human serotransferrin, ovalbumin and contaminations of two other: bovine alpha 1 acid glycoprotein (bAGP) and bovine fetuin.

View Article: PubMed Central - PubMed

Affiliation: Thermo Fisher Scientific, San Jose, CA 95134, USA.

ABSTRACT
Currently, glycans are attracting attention from the scientific community as potential biomarkers or as posttranslational modifications (PTMs) of therapeutic proteins. However, structural characterization of glycoproteins and glycopeptides remains analytically challenging. Here, we report on the implementation of a novel acquisition strategy termed higher-energy collision dissociation-accurate mass-product-dependent electron transfer dissociation (HCD-PD-ETD) on a hybrid linear ion trap-orbitrap mass spectrometer. This acquisition strategy uses the complementary fragmentations of ETD and HCD for glycopeptides analysis in an intelligent fashion. Furthermore, the approach minimizes user input for optimizing instrumental parameters and enables straightforward detection of glycopeptides. ETD spectra are only acquired when glycan oxonium ions from MS/MS HCD are detected. The advantage of this approach is that it streamlines data analysis and improves dynamic range and duty cycle. Here, we present the benefits of HCD-PD-ETD relative to the traditional alternating HCD/ETD for a trainer set containing twelve-protein mixture with two glycoproteins: human serotransferrin, ovalbumin and contaminations of two other: bovine alpha 1 acid glycoprotein (bAGP) and bovine fetuin.

No MeSH data available.


Related in: MedlinePlus