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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

Schematic representation of HCD-PD-ETD acquisition method.
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Related In: Results  -  Collection


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fig1: Schematic representation of HCD-PD-ETD acquisition method.

Mentions: A Thermo Scientific EASY-nLC nano-HPLC system and Michrom Magic C18 spray tip 15 cm × 75 μm i.d. column (Auburn, CA) were used. Gradient elution was performed from 5 to 45% ACN in 0.1% formic acid over 60 min at a flow rate of 300 nL/min. The samples were analyzed with a Thermo Scientific LTQ Orbitrap Velos hybrid mass spectrometer with ETD. The following MS and MS/MS settings were used: FT: MSn AGC Target = 5e4; MS/MS = 1 μscans, 200 ms max ion time; MS = 400–2000 m/z, 60000 resolution at m/z 400, MS Target = 1e6; MS/MS = Top 10 data-dependent acquisition HCD product-dependent acquisition ion trap ETD (Figure 1), dynamic exclusion = repeat count 1, Duration 30 sec, exclusion duration 90 sec; HCD Parameters: collision energy = 35%; resolution 7500. MSn target ion trap = 1e4, 3 μscans, 150 ms max ion time; ETD anion AGC target = 2e5, and charge-dependent ETD reaction time was used.


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)

Schematic representation of HCD-PD-ETD acquisition method.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Schematic representation of HCD-PD-ETD acquisition method.
Mentions: A Thermo Scientific EASY-nLC nano-HPLC system and Michrom Magic C18 spray tip 15 cm × 75 μm i.d. column (Auburn, CA) were used. Gradient elution was performed from 5 to 45% ACN in 0.1% formic acid over 60 min at a flow rate of 300 nL/min. The samples were analyzed with a Thermo Scientific LTQ Orbitrap Velos hybrid mass spectrometer with ETD. The following MS and MS/MS settings were used: FT: MSn AGC Target = 5e4; MS/MS = 1 μscans, 200 ms max ion time; MS = 400–2000 m/z, 60000 resolution at m/z 400, MS Target = 1e6; MS/MS = Top 10 data-dependent acquisition HCD product-dependent acquisition ion trap ETD (Figure 1), dynamic exclusion = repeat count 1, Duration 30 sec, exclusion duration 90 sec; HCD Parameters: collision energy = 35%; resolution 7500. MSn target ion trap = 1e4, 3 μscans, 150 ms max ion time; ETD anion AGC target = 2e5, and charge-dependent ETD reaction time was used.

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