Method to identify the glycosidic linkage of sialic acid in glycopeptides

A method for identifying one or more linkages of a sialic acid (SA) to a glycan of an isomer of a glycopeptide using electron capture dissociation (ECD) and multiple reaction monitoring (MRM) precursor ion to product ion transitions, comprising:

• calculating a separation time of an isomer of one or more isomers of a glycopeptide of a sample from a peak of an extracted ion chromatogram (XIC) using a processor, wherein the one or more isomers are separated from the sample using a separation device, the one or more separated isomers are ionized using an ion source, producing an ion beam that includes isomer ions of a precursor ion of the glycopeptide, and
• for each separation time of a plurality separation times, using an ECD device with 2-5 eV electrons, a first group of one or more MRM transitions that each includes the precursor ion and a product ion known to be enhanced or suppressed for a first linkage of an SA of the glycoprotein to a glycan of the glycopeptide and a second group of one or more MRM transitions that each includes the precursor ion and a product ion known to be enhanced or suppressed for a second linkage for the SA to the glycan are executed on the ion beam, producing the XIC for the precursor ion and an XIC for each product ion of the first and second groups;
• summing product ion intensities of the first group at the separation time producing a first sum and summing product ion intensities of the second group at the separation time producing a second sum using XICs of the first and second groups using the processor;
• calculating a ratio of the first sum to the second sum using the processor;
• comparing the ratio at the separation time to predetermined ratio ranges that each corresponds to a combination of a selection from a set of the first linkage and the second linkage taken one or more times using the processor, wherein one or more times correspond to one or more SAs known to be included in the glycopeptide;
• and
• identifying one or more linkages of the SA to the glycan of the isomer from a combination found to match the ratio in the comparison using the processor.

A computer program product, comprising a non-transitory and tangible computer-readable storage medium whose contents include a program with instructions being executed on a processor to perform a method for identifying one or more linkages of a sialic acid (SA) to a glycan of an isomer of a glycopeptide using electron capture dissociation (ECD) and multiple reaction monitoring (MRM) precursor ion to production transitions, the method comprising:

• providing a system, wherein the system comprises one or more distinct software modules, and wherein the distinct software modules comprise an analysis module;
• calculating a separation time of an isomer of one or more isomers of a glycopeptide of a sample from a peak of an XIC using the analysis module, wherein the one or more isomers are separated from the sample using a separation device, the one or more separated isomers are ionized using an ion source, producing an ion beam that includes isomer ions of a precursor ion of the glycopeptide, and for each separation time of a plurality separation times, using an ECD device with 2-5 eV electrons, a first group of one or more MRM transitions that each includes the precursor ion and a production known to be enhanced or suppressed for a first linkage of an SA of the glycoprotein to a glycan of the glycopeptide and a second group of one or more MRM transitions that each includes the precursor ion and a product ion known to be enhanced or suppressed for a second linkage for the SA to the glycan are executed on the ion beam, producing the XIC for the precursor ion and an XIC for each product ion of the first and second groups;
• summing product ion intensities of the first group at the separation time producing a first sum and summing product ion intensities of the second group at the separation time producing a second sum using XICs of the first and second groups using the analysis module;
• calculating a ratio of the first sum to the second sum using the analysis module;
• comparing the ratio at the separation time to predetermined ratio ranges that each corresponds to a combination of a selection from a set of the first linkage and the second linkage taken one or more times using the analysis module, wherein one or more times correspond to one or more SAs known to be included in the glycopeptide;
• and
• identifying one or more linkages of the SA to the glycan of the isomer from a combination found to match the ratio in the comparison using the analysis module.