In this application note an Agilent ZORBAX Rapid Resolution High Definition (RRHD) 300-HILIC 1.8 ?m column and an Agilent AdvanceBio Peptide Mapping column are used in combination with time of flight (TOF) mass-spectrometry (MS) for mapping EPO protein. The work demonstrates the utility of HILIC as an orthogonal and complementary approach to reversed-phase LC/MS for peptide analysis.
In this work, an AdvanceBio Peptide Mapping column is used to generate a rapid and highly efficient peptide map at a traditional LC system pressure. The column achieves substantial improvements in peptide mapping during very fast run times and low system pressures, while still maintaining high peak-performance efficiency.
Reversed-phase UHPLC/HPLC is routinely used for peptide mapping, but if the digest contains hydrophilic peptides, such as glycopeptides, valuable information can be missed. This note demonstrates peptide mapping of digested glycoprotein erythropoietin (EPO) protein using an Agilent ZORBAX Rapid Resolution High Definition 300-HILIC 1.8 ?m LC column and a TOF MS. Taking advantage of the high organic solvent system of the mobile phase for HILIC (hydrophilic interaction chromatography), the glycopeptides are effectively dissolved, retained and separated by the HILIC column. This app note includes the separation, sequence coverage and comparison of reversed-phase and HILIC data to demonstrate the utility of HILIC as an orthogonal and complementary approach to RPLC/MS for peptide analysis.
In this note, we describe a fast and efficient liquid chromatography-TOF mass spectrometry approach to characterize an IgG derived from CHO cells. An Agilent Poroshell 300SB-C3, 5 um superficially porous IgG1 column was used to obtain accurate glycoform masses of an intact IgG1, with a subsequent analysis of papain-digeste3d IgG1 to obtain site-specific glycosylation profile information of the Fc region for further characterization of IgG1 heterogeneity. Additionally, a subsequent mAb-glyco chip LC/MS analysis was used to build a glycan accurate mass database for additional validation of the glycans identified in the TOF mass analysis.