Enhanced glyco‐profiling by specific glycopeptide enrichment and complementary monolithic nano‐LC (ZIC‐HILIC/RP18e)/ESI‐MS analysis

Abstract

Dedicated and specific sample preparation and adequate chromatographic resolution prior to MS are necessary for comprehensive and site-specific glycosylation analysis to compensate for high heterogeneity of protein glycosylation, low-abundance of specific glycoforms and ion-suppression effects caused by coelution of other peptides. This article describes a scheme for glycopeptide profiling, which comprises HILIC batch enrichment followed by complementary HILIC and RP-LC in 1-D and 2-D approaches. For reproducible and sensitive nano-LC/ESI-MS analysis, we used ZIC-HILIC and RP18e monolithic silica capillaries and assessed their retention characteristics and complementarity for glycopeptide separations. The experiments revealed that pre-enrichment of glycopeptides in combination with LC employing both phases considerably improves site-specific elucidation of glycosylation heterogeneity. Zwitterionic hydrophilic interaction liquid chromatography showed high capability to separate glycopeptides by their glycan composition, which coeluted on RP18e. By varying solvent conditions, retention can be well tuned, and efficient separations were achieved even in absence of any additives like salt or formic acid. RP18e facilitated glycopeptide separations with high peak capacity based on peptide sequence and degree of sialylation. Implementing both orthogonal and complementary phases in 1-D and 2-D LC setups was shown to significantly increase the number of different identified glycoforms and possesses great potential for comprehensive glycoproteomics approaches.

Optimization of In Vitro N-Deglycosylation of Ovomucoid Protein

“…In fact, we have found three new Asn at position 47, 190 and 193, that are probably glycosylated. Several peptide sequences have been identified as demonstrated by the yield of different identified glycopeptides ( Table 2 summarizes the results of this study) confirming the high site-specific glycosylation heterogeneity [31,32] existing within the ovomucoid molecule.…”

Direct and Indirect Applications of Sub- and Supercritical Water in Food-Related Analysis

“…The mechanism of analyte retention in HILIC involves the acidobasic proton donor-acceptor and dipole-dipole interactions of the analyte with both the surface of the stationary phase and with the liquid (mostly water) occluded on the surface in the diffusion layer. Monolithic silica can be turned into a HILIC stationary phase through modification of the monolith surface with a suitable organic moiety (Ikegami et al , 2008Horie et al 2007;Malerod et al 2013), e.g., with a zwitterionic sulfobetaine (Wohlgemuth et al 2010). An example of the monolith modification (Moravcová et al 2012) involves a two-step process.…”

Theory and Practice of UHPLC and UHPLC–MS

“…HILIC on a capillary zwitterionic ZIC-cHILIC column in combination with electrospray ionization mass spectrometry has been applied to the detection and identification of more than 100 N-glycopeptides and O-glycopeptides in a single run (Takegawa et al, 2008). Glycopeptides could be distinguished by their glycan composition on a monolithic silica gel capillary surface modified with sulfobetaine stationary phase, in contrast to RP-HPLC (Wohlgemuth et al, 2010). Purine and pyrimidine bases and nucleosides can be separated using gradient elution with a decreasing concentration of acetonitrile in buffered aqueouseorganic mobile phase on sulfobetaine ZIC-HILIC columns.…”