DIFFERENTIATING CARBOHYDRATE STRUCTURAL AND … · structural and positional carbohydrate is...
1
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2011 Waters Corporation FRAGMENTATION FOLLOWING IMS SEPARATION BY SYNAPT G2 HDMS OVERVIEW A sensitive method, based on travelling wave ion-mobility mass spectrometry (TWIMS), for the rapid characterization of structural and positional carbohydrate is presented Structural and positional isomers are separated by travelling wave ion-mobility and subsequently identified by fragmentation spectra, obtained in the transfer cell of the Synapt G2 mass spectrometer, providing more confident structural information The collisional cross section (CCS) values of the separated isomers were automatically calculated after the system was calibrated using a polyalanine calibration standard MATERIALS & METHODS Samples: Oligosaccharide standards were purchased from commercial sources, and were dissolved in a mixture of water/methanol (1:1; v/v) doped with sodium acetate (positive ion) or ammonium phosphate (negative ion) to a concentration of 10 pmol/μL. N-linked glycans from 100 μg of chicken ovalbumin (grade VI), were released by PNGase overnight. The isolated glycans were lyophilized and re-suspended in 50% (v/v) methanol/aqueous solution for MS analysis. Based on protein concentration, the concentration of total glycan is estimated ~ 2 pmol/uL. Sample Introduction MALDI MS analysis: the glycan standards were mixed 1:1 (v/v) with a 2,5-Dihydroxybenzoinc acid (DHB) MALDI matrix solution (20 mg/mL in ethanol), before spotting onto a stainless steel MALDI target plate for analysis. For electrospray ionization analysis of glycans, sample introduction was achieved by a direct infusion at a flow rate of 5 μL/min. Ion mobility—Mass spectrometry All the analysis was performed on a Synapt G2 HDMS mass spectrometer equipped with either a MALDI or a ESI source. The instrument was operated at a source temperature of 105 ◦ C. The cone voltage and the collision energy was optimized separately to achieve the best MS signal or suitable fragmentation spectra for each of the glycans analyzed. Typically the cone was set at high voltage (between 60 and 105 V) for ESI-MS/MS experiments to obtain strong sodiated signals. Mobility experiments were performed with a fixed wave height of 40 V (positive ion) and 16 V (negative ion) at the IMS gas pressure of 3.0 mbar. The velocity is adjusted to achieve optimized separation for different glycans. Informatics & Collisional Cross-section Determination Collisional cross-section of each oligosaccharide was automatically calculated after the system was calibrated using a polyalanine calibration standard using DriftScope. (see right for details) DIFFERENTIATING CARBOHYDRATE STRUCTURAL AND POSITIONAL ISOMERS BY ION-MOBILITY MASS SPECTROMETRY Weibin Chen, Ying-Qing Yu, Asish Chakraborty, Henry Shion, StJohn Skilton Biopharmaceutical Sciences, Waters Corporation, 34 Maple Street, Milford, MA 01757 CONCLUSIONS Travelling-wave ion mobility mass spectrometry is a rapid and efficient technique for the differentiation of carbohydrate structural and positional isomers. Collisional cross sectional areas of oligosaccharides can be readily obtained by following an automatic routine in DriftScope. These oligonucleotides can be described not only by their mass or mass- to-charge but by their collisional cross-sectional areas. The Triwave technology of the Synapt G2 HDMS mass spectrometer offers enhanced functionality by performing fragmentation either before and/or after ion mobility separation to elucidate the complex structures of oligosaccharides. The positive ion electrospray analysis of a sample of Ovalbumin glycans released with PNGase F overnight without any prior sample cleanup. The data is displayed in the Driftscope (Figure 1). Two bands of singly and doubly charged glycan ions were present along with the multiple bands of ions from contaminants in the sample. The N-glycans were separated into the region marked with the ovals. The spectra of the ions falling in this region is shown in Figure 2. The whole range of N-glycans can now be clearly seen. Figure 2C shows the N-glycan ions at higher mass that are completely obscured by contaminating ions in the original ESI spectrum are now clearly displayed after the removal of the signals from contaminants. An Automatic Work Routine for the Determination of Collisional- Cross Section of Oligosaccharides after IMS Separation Results and Discussion Differentiating Structural Isomers from Released Glycans of Ovalbumin Figure 4. Positive ion MS/MS spectra of the first and second N- glycan isomers of m/z 1136 from chicken ovalbumin Figure 5. Differentiating structural oligosaccharide isomers from human milk using MALDI Synapt G2 HDMS Differentiating Carbohydrate Positional Isomers WATERS SYNAPT G2 HDMS Carbohydrate Isomers m/z Drift Time TOF Analyzer Drift time m/z 20110524_UCA064_WBC_OVALGLYCAN_12.raw : 1 Region 1 Region 2 Figure 1. Ion mobility separation of N-glycans (circled) from contaminating species in the Ovalbumin sample. The summed ESI spectrum is shown in Figure 2A. Figure 2. Positive ion spectra of ovalbumin glycans extracted by ion mobility. (A) Mass spectrum from all the species in the sample in the range of m/z: 700—1800 (B) Mass spectrum for the released glycans from ovalbumin after IMS separation (C) Mass spectrum of released glycans from Region 1 in Figure 1 (D) Mass spectrum of released glycans from Region 2 in Figure 2. D C B A Raw Data of CCS Calibrant Autopeak Detection for Calibrant Set up CCS Calibration Plot Apply the Calib. Plot to Analyte Figure 3. Positive ion mass spectrum obtained for glycans released from ovalbumin using the Synapt G2 HDMS instrument. The peaks for sodiated (GlcNAc) n Man 3 GlcNAc 2 (n=0,1,2) are labeled with their respective drift times. m/z: 933.3 m/z: 1136.3 m/z: 1339.3 B 1 B 1 H 2 Na H 3 Na H 2 N 1 N B 3 B 4 C 4 0,2 A 5 [M+Na] + H 2 Na H 3 Na H 2 N 1 N B 3 B 4 C 4 0,2 A 5 [M+Na] + REFERENCES L. S. Fenn et al. Phys. Chem. Chem. Phys., 2011, 13, 2196-2205 J. P. Williams et al. Int. J. Mass Spectrom., 2010, 298, 119-127 D. J. Harvey, et al. J. Am. Soc. Mass Spectrom. 2000, 11, 564-571 M. D. Plasencia, et al. J. Am. Soc. Mass Spectrom. 2008, 19, 1706-1715 Glc 1-6Glc 1-4Glc Glcβ1-4Glcβ1-4Glc R R NeuNAc 2-3Gal 1-4Glc NeuNAc 2-6Gal 1-4Glc Isomer Mixture Figure 6. Structures of the positional isomers and the corresponding drift time profiles. The difference between each pairs of isomers only takes place at the linkage variations.
Transcript of DIFFERENTIATING CARBOHYDRATE STRUCTURAL AND … · structural and positional carbohydrate is...
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2011 Waters Corporation
FRAGMENTATION FOLLOWING IMS SEPARATION BY SYNAPT G2 HDMS
OVERVIEW A sensitive method, based on travelling wave ion-mobility mass
spectrometry (TWIMS), for the rapid characterization of structural and positional carbohydrate is presented
Structural and positional isomers are separated by travelling wave ion-mobility and subsequently identified by fragmentation spectra, obtained in the transfer cell of the Synapt G2 mass spectrometer, providing more confident structural information
The collisional cross section (CCS) values of the separated isomers were automatically calculated after the system was calibrated using a polyalanine calibration standard
MATERIALS & METHODS
Samples:
Oligosaccharide standards were purchased from commercial sources, and were dissolved in a mixture of water/methanol (1:1; v/v) doped with sodium acetate (positive ion) or ammonium phosphate (negative ion) to a concentration of 10 pmol/µL.
N-linked glycans from 100 µg of chicken ovalbumin (grade VI), were released by PNGase overnight. The isolated glycans were lyophilized and re-suspended in 50% (v/v) methanol/aqueous solution for MS analysis. Based on protein concentration, the concentration of total glycan is estimated ~ 2 pmol/uL.
Sample Introduction
MALDI MS analysis: the glycan standards were mixed 1:1 (v/v) with a 2,5-Dihydroxybenzoinc acid (DHB) MALDI matrix solution (20 mg/mL in ethanol), before spotting onto a stainless steel MALDI target plate for analysis.
For electrospray ionization analysis of glycans, sample introduction was achieved by a direct infusion at a flow rate of 5 µL/min.
Ion mobility—Mass spectrometry
All the analysis was performed on a Synapt G2 HDMS mass spectrometer equipped with either a MALDI or a ESI source. The instrument was operated at a source temperature of 105◦C. The cone voltage and the collision energy was optimized separately to achieve the best MS signal or suitable fragmentation spectra for each of the glycans analyzed. Typically the cone was set at high voltage (between 60 and 105 V) for ESI-MS/MS experiments to obtain strong sodiated signals. Mobility experiments were performed with a fixed wave height of 40 V (positive ion) and 16 V (negative ion) at the IMS gas pressure of 3.0 mbar. The velocity is adjusted to achieve optimized separation for different glycans.
Informatics & Collisional Cross-section Determination
Collisional cross-section of each oligosaccharide was automatically calculated after the system was calibrated using a polyalanine calibration standard using DriftScope. (see right for details)
DIFFERENTIATING CARBOHYDRATE STRUCTURAL AND POSITIONAL ISOMERS BY ION-MOBILITY MASS SPECTROMETRY Weibin Chen, Ying-Qing Yu, Asish Chakraborty, Henry Shion, StJohn Skilton Biopharmaceutical Sciences, Waters Corporation, 34 Maple Street, Milford, MA 01757
CONCLUSIONS Travelling-wave ion mobility mass spectrometry is a rapid and
efficient technique for the differentiation of carbohydrate structural and positional isomers.
Collisional cross sectional areas of oligosaccharides can be readily obtained by following an automatic routine in DriftScope. These oligonucleotides can be described not only by their mass or mass-to-charge but by their collisional cross-sectional areas.
The Triwave technology of the Synapt G2 HDMS mass spectrometer offers enhanced functionality by performing fragmentation either before and/or after ion mobility separation to elucidate the complex structures of oligosaccharides.
The positive ion electrospray analysis of a sample of Ovalbumin glycans released with PNGase F overnight without any prior sample cleanup. The data is displayed in the Driftscope (Figure 1). Two bands of singly and doubly charged glycan ions were present along with the multiple bands of ions from contaminants in the sample. The N-glycans were separated into the region marked with the ovals. The spectra of the ions falling in this region is shown in Figure 2. The whole range of N-glycans can now be clearly seen. Figure 2C shows the N-glycan ions at higher mass that are completely obscured by contaminating ions in the original ESI spectrum are now clearly displayed after the removal of the signals from contaminants.
An Automatic Work Routine for the Determination of Collisional-Cross Section of Oligosaccharides after IMS Separation
Results and Discussion Differentiating Structural Isomers from Released Glycans
of Ovalbumin
Figure 4. Positive ion MS/MS spectra of the first and second N-glycan isomers of m/z 1136 from chicken ovalbumin
Figure 5. Differentiating structural oligosaccharide isomers from human milk using MALDI Synapt G2 HDMS
Differentiating Carbohydrate Positional Isomers WATERS SYNAPT G2 HDMS
Carbohydrate Isomers
m/z
Drift Time
TOF
An
alyz
er
Drift time
m/z
20110524_UCA064_WBC_OVALGLYCAN_12.raw : 1
Region 1
Region 2
Figure 1. Ion mobility separation of N-glycans (circled) from contaminating species in the Ovalbumin sample. The summed ESI spectrum is shown in Figure 2A.
Figure 2. Positive ion spectra of ovalbumin glycans extracted by ion mobility. (A) Mass spectrum from all the species in the sample in the range of m/z: 700—1800 (B) Mass spectrum for the released glycans from ovalbumin after IMS separation (C) Mass spectrum of released glycans from Region 1 in Figure 1 (D) Mass spectrum of released glycans from Region 2 in Figure 2.
D
C
B
A Raw Data of CCS Calibrant Autopeak Detection for Calibrant
Set up CCS Calibration Plot Apply the Calib. Plot to Analyte
Figure 3. Positive ion mass spectrum obtained for glycans released from ovalbumin using the Synapt G2 HDMS instrument. The peaks for sodiated (GlcNAc)nMan3GlcNAc2 (n=0,1,2) are labeled with their respective drift times.
m/z: 933.3
m/z: 1136.3
m/z: 1339.3
B1
B1
H2Na H3Na H2N1N
B3 B4
C4 0,2A5
[M+Na]+
H2Na H3Na
H2N1NB3
B4
C4 0,2A5
[M+Na]+
REFERENCES
L. S. Fenn et al. Phys. Chem. Chem. Phys., 2011, 13, 2196-2205 J. P. Williams et al. Int. J. Mass Spectrom., 2010, 298, 119-127 D. J. Harvey, et al. J. Am. Soc. Mass Spectrom. 2000, 11, 564-571 M. D. Plasencia, et al. J. Am. Soc. Mass Spectrom. 2008, 19, 1706-1715
Glc 1-6Glc 1-4Glc
Glcβ1-4Glcβ1-4Glc
R
R
NeuNAc 2-3Gal 1-4Glc
NeuNAc 2-6Gal 1-4Glc
Isomer Mixture
Figure 6. Structures of the positional isomers and the corresponding drift time profiles. The difference between each pairs of isomers only takes place at the linkage variations.
