It is all about the solvent: on the importance of the mobile phase for ZIC-HILIC glycopeptide enrichment

Abstract

Glycopeptide enrichment is a crucial step in glycoproteomics for which hydrophilic interaction chromatography (HILIC) has extensively been applied due to its low bias towards different glycan types. A systematic evaluation of applicable HILIC mobile phases on glycopeptide enrichment efficiency and selectivity is, to date, however, still lacking. Here, we present a novel, simplified technique for HILIC enrichment termed “Drop-HILIC”, which was applied to systematically evaluate the mobile phase effect on ZIC-HILIC (zwitterionic type of hydrophilic interaction chromatography) glycopeptide enrichment. The four most commonly used MS compatible organic solvents were investigated: (i) acetonitrile, (ii) methanol, (iii) ethanol and (iv) isopropanol. Glycopeptide enrichment efficiencies were evaluated for each solvent system using samples of increasing complexity ranging from well-defined synthetic glycopeptides spiked into different concentrations of tryptic BSA peptides, followed by standard glycoproteins, and a complex sample derived from human (depleted and non-depleted) serum. ZIC-HILIC glycopeptide efficiency largely relied upon the used solvent. Different organic mobile phases enriched distinct glycopeptide subsets in a peptide backbone hydrophilicity-dependant manner. Acetonitrile provided the best compromise for the retention of both hydrophilic and hydrophobic glycopeptides, whereas methanol was confirmed to be unsuitable for this purpose. The enrichment efficiency of ethanol and isopropanol towards highly hydrophobic glycopeptides was compromised as considerable co-enrichment of unmodified peptides occurred, though for some hydrophobic glycopeptides isopropanol showed the best enrichment properties. This study shows that even minor differences in the peptide backbone and solvent do significantly influence HILIC glycopeptide enrichment and need to be carefully considered when employed for glycopeptide enrichment. Graphical AbstractThe organic solvent plays a crucial role in ZIC-HILIC glycopeptide enrichment Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-016-0051-6) contains supplementary material, which is available to authorized users.

Nano reversed phase versus nano hydrophilic interaction liquid chromatography on a chip in the analysis of hemopexin glycopeptides

“…HILIC is suitable for the analysis of polar analytes which are inadequately retained in RP-LC [1]. HILIC was successfully applied to glycopeptide enrichment [7,8], released glycan analysis [9,10], and separation of different glycoforms of peptides [11]. Although the directed analysis of glycoproteins provides site-specific information on glycans, the common approach to characterize glycans in HILIC is their enzymatic or chemical releas LC/MS analysis [12].…”

Hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs and therapeutic peptides: A review based on the separation characteristics of the hydrophilic interaction chromatography phases

“…Similar glycopeptides from IgG1, IgG2, and IgG4 were finely separated on Glycan BEH Amide , TSKgel Amide‐80 , and Halo Penta‐HILIC . Sialylated glycopeptides on EEQYNSTYR and EEQFNSTFR were analyzed by Drop‐HILIC enrichment, followed by RPLC–ESI–MS/MS . The EEQYNSTYR glycopeptides is highly polar and hydrophilic, so completely aqueous conditions are required to trap it on RP phases.…”

To enrich or not to enrich: Enhancing (glyco)peptide ionization using the CaptiveSpray nanoBooster™

“…Hydrophilic Interaction Chromatography (HILIC) has extensively been applied for this purpose due to its low bias towards different glycan types. However, a recent systematic evaluation of the mobile phase effect on glycopeptide enrichment indicated that zwitterionic (ZIC)-HILIC glycopeptide efficiency primarily relied upon the used solvent 25 . Thus, if the analytical conditions allow the detection of glycopeptides from complex samples without any enrichment steps, any enrichment-derived bias would be significantly reduced.…”

“…More importantly, glycopeptide enrichment efficiency was strongly dependent on the used organic phase 25 Figure S3; Figure 2 and Supplementary Table S4 and S5). Glycopeptides were generally detected in higher charge states when ACN was used as the dopant solvent.…”