Qiuting Hong1; L. Renee Ruhaak1; Suzanne Miyamoto2; Carlito Lebrilla1
1Chemsitry, UC, Davis, Davis, CA; 2Comprehensive Cancer Center, UC, Davis, Davis, CA
NOVEL ASPECT: Monitoring the degree of glycosylation with the absolute protein quantification using MRM methods
INTRODUCTION:
The studies aimed towards glycan biomarker discovery have focused on glycan characterization and profiling of released glycans. Site-specific glycosylation analysis is less developed but may provide a new type of biomarkers with higher sensitivity and specificity. Quantitation of peptide-conjugated glycans directly facilitates the differential analysis of distinct glycoforms associated with specific proteins at distinct sites. We have developed a method using MRM to monitor protein glycosylation normalized to absolute protein concentrations to examine quantitative changes in glycosylation at a site-specific level. The remarkable sensitivity and selectivity of MRM have enabled the detection of low abundant glycopeptides from serum directly.
The studies aimed towards glycan biomarker discovery have focused on glycan characterization and profiling of released glycans. Site-specific glycosylation analysis is less developed but may provide a new type of biomarkers with higher sensitivity and specificity. Quantitation of peptide-conjugated glycans directly facilitates the differential analysis of distinct glycoforms associated with specific proteins at distinct sites. We have developed a method using MRM to monitor protein glycosylation normalized to absolute protein concentrations to examine quantitative changes in glycosylation at a site-specific level. The remarkable sensitivity and selectivity of MRM have enabled the detection of low abundant glycopeptides from serum directly.
METHODS:
The glycosylation pattern of protein standards was first determined using both trypsin and pronase digestion. Proteins were treated with DTT and IAA before an overnight digestion in a 37°C water bath. Glycopeptides were profiled using an Agilent 6520 HPLC-Chip/QTOF MS, and identified using an in-house-software tool, GPFinder. Once the site specific glycosylation was determined, quantification was performed with the tryptic peptides using an Agilent 6490 QqQ MS coupled with an Agilent 1290 UPLC system. The absolute amount of proteins was determined by the peak area of peptides, while the degree of glycosylation was normalized to the protein content, thus allowing quantification of glycoforms on the site-specific and protein-specific level. The method was applied to glycoproteins in 11 healthy human sera.
The glycosylation pattern of protein standards was first determined using both trypsin and pronase digestion. Proteins were treated with DTT and IAA before an overnight digestion in a 37°C water bath. Glycopeptides were profiled using an Agilent 6520 HPLC-Chip/QTOF MS, and identified using an in-house-software tool, GPFinder. Once the site specific glycosylation was determined, quantification was performed with the tryptic peptides using an Agilent 6490 QqQ MS coupled with an Agilent 1290 UPLC system. The absolute amount of proteins was determined by the peak area of peptides, while the degree of glycosylation was normalized to the protein content, thus allowing quantification of glycoforms on the site-specific and protein-specific level. The method was applied to glycoproteins in 11 healthy human sera.
ABSTRACT:
We determined the site-specific glycosylation of immunoglobulin G, A, and M, transferrin, alpha-2-macroglobulin, and haptoglobin using nonspecific proteolysis. Tryptic digestion yielded limited information compared to nonspecific protease analysis, but was necessary to obtain a consistent peptide sequence for MRM.
We determined the site-specific glycosylation of immunoglobulin G, A, and M, transferrin, alpha-2-macroglobulin, and haptoglobin using nonspecific proteolysis. Tryptic digestion yielded limited information compared to nonspecific protease analysis, but was necessary to obtain a consistent peptide sequence for MRM.
The CID fragmentation of the tryptic glycopeptides was determined to find the optimal transition for the MRM experiments. Quantitation experiments were performed on a triple quadrupole optimized for glycopeptide analysis with MRM transitions from molecular ions to either m/z 203 (HexNAc) or m/z 366 (HexNAc-Hex).
MRM is not typically used for glycopeptide quantitation. The problem is the lack of glycopeptide standards. In this research, we use the absolute protein abundances to normalize the glycopeptide abundances. In this way, relative changes in protein glycosylation can be measured at the site-specific and protein-specific level.
As an example, a peptide that is common to IgG1, IgG2, IgG3 and IgG4 was quantified to obtain the absolute amount of IgG protein. The result showed a wide dynamic range (>1000) and low LOD (6.01fmole). The average IgG concentration for a set of 11 samples was 5.25mg/ml, with a relative standard deviation (RSD) of 21.8%. Peptides that were unique for each subclass were quantified to monitor the variations in abundances for the four IgG subclasses.
As an example, a peptide that is common to IgG1, IgG2, IgG3 and IgG4 was quantified to obtain the absolute amount of IgG protein. The result showed a wide dynamic range (>1000) and low LOD (6.01fmole). The average IgG concentration for a set of 11 samples was 5.25mg/ml, with a relative standard deviation (RSD) of 21.8%. Peptides that were unique for each subclass were quantified to monitor the variations in abundances for the four IgG subclasses.
Using this method, we showed that specific glycoforms can be monitored for IgG (25 tryptic glycopeptides), transferrin (3), IgA (6), alpha-2-macroglobulin (5), and haptoglobin (3) directly from serum. Some sites from some proteins were not observed due to the problems associated with tryptic digestion of glycoproteins. Our normalized glycopeptide abundances, which remove the contribution of protein concentration, gave results allowed the simultaneous monitoring of glycosylation across several proteins and several sites. This method will have implications in the discovery of glycan biomarker by associating the glycans to specific proteins.