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Glycan quantification using external standards
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Glycan quantification using external standards

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Introduction Protocol References Credit lines
Category
Glyco-proteomic mass spectrometry protocols
Protocol Name

Glycan quantification using external standards

Authors
 Porterfield, Melody
Complex Carbohydrate Research Center, University of Georgia

Aoki, Kazuhiro
Complex Carbohydrate Research Center, University of Georgia

Tiemeyer, Michael *
Complex Carbohydrate Research Center, University of Georgia
*To whom correspondence should be addressed.
KeyWords
Reagents
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Malto oligosaccharides kit (Dp3 ~ Dp7), (Supelco/Sigma-Aldrich, St. Louis, MO) or other well-characterized oligosaccharide standards available

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Anhydrous methanol, 99.8%, (Sigma-Aldrich, St. Louis, MO)

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Methanol, HPLC, (Sigma-Aldrich)

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50% sodium hydroxide (Thermo Fisher Scientific Inc., Waltham, MA)

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Deionized water

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Iodemethane-13C, 99atom % 13C, (Sigma-Aldrich)

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Anhydrous dimethyl sulfoxide, ≥99.9% (Sigma-Aldrich)

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Infusion buffer, 1mM NaOH in 50% MeOH

Instruments
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Vortex mixer
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Glass tube with screw top and cap
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Centrifuge, benchtop
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Glass pipettes
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Nitrogen evaporator
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LTQ-Orbitrap Discoverer (Thermo Fisher Scientific Inc., Waltham, MA)
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Or any mass spectrometer
Methods
1.

Preparation of Standards

1) 

 A mixture of oligosaccharide standards (e.g. Dp3 and Dp4) is permethylated with 13C-MeI and dried under nitrogen stream according to a method of Anumula and Taylor (1)

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2) 

 Permethylated oligosaccharide standards mixture is resuspended in MeOH and stored at −20ºC in a screw-top glass tube until use.

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3) 

 An aliquot of a known concentration of permethylated oligosaccharide standard is added to infusion buffer.

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2.

Standard curve

1) 

 To determine the concentrations to make a linear calibration curve for your instrument, a mixture of permethylated oligosaccharide standards is diluted and several concentrations of the standard are infused into the mass spectrometer.

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2) 

 The signal intensities are used to make the calibration curve.

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3) 

 Use concentrations that demonstrate linear regression.

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3.

Sample analysis

1) 

 A full MS profile of the unknown concentration of permethylated glycan is taken to determine the appropriate amount of standard to add.

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2) 

 A known amount of standard is then mixed with the glycan sample.

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3) 

 A mixture of standard and glycan sample is analyzed by MS spec.

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4) 

 All intensities are reported as relative to the spiked external standard.

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Notes
  1. The standard(s) selected should not have the same mass as any peaks in the unknown sample.  Therefore a full MS profile of your sample must be taken prior to quantitative analysis to select which standard to use. 
  2. Standards are heavy permethylated using 13C-MeI, deuterated iodomethane (CD3I) or 13CD3I to avoid coinciding with the mass of hexose ladder that may be present in the unknown samples. 
Figure & Legends

Figure & Legends

 

 

 

Fig. 1.  Structure and mass of 13C-labeled Dp4

 

 

 

 

Fig. 2.  Full MS profile of permethylated N-glycan from human plasma analyzed with 13C-labeled Dp3 and Dp4 STDs.

 

 

 

Fig. 3.  Calibration curve of permethylated Dp4 demonstrates linear regression across a range of xx pmol ~ xx nmol.

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