Sensitive and fast characterization of site-specific protein glycosylation with capillary electrophoresis coupled to mass spectrometry

Abstract

Glycoproteomic analysis requires efficient separation and sensitive detection to enable the comprehensive characterization of glycan heterogeneity. Here, we report the use of capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) with an electrokinetically-pumped nanospray interface for the study of protein glycosylation microheterogeneity. A fast separation was developed that resolved intact glycopeptides generated from standard proteins within ~9min. Differentially terminal-galactosylated and sialylated species with the same glycosylation sites were well resolved. The concentration detection limits for CZE were three times higher than for nanoLC methods; however, a 200-fold smaller injection volume was used in CZE, which reflects the use of an extremely efficient electrospray interface in our CZE-ESI-MS setup. The resulting glycopeptide mass detection limit was two orders of magnitude superior to a nanoLC method. We also observed a 1.5% and 7% average relative standard deviation in peak migration time and glycopeptide relative abundance, and a four order of magnitude linear dynamic range in signal intensity. With CZE-ESI-MS, 40 haptoglobin glycopeptides were identified from roughly 40 fmol of digest.

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation, methodology and applications

“…Later, a glycosylation assay for PSA analysis in urine was developed by the same group, based on a similar CE method in combination with dopant (ACN) enriched nitrogen gas to enhance sensitivity . In addition, the Dovichi group observed an improved resolution of different N ‐glycopeptide species with the same peptide sequence, especially if differentially sialylated, compared to RPLC . In a subsequent study, the same group demonstrated an offline RPLC‐CZE‐ESI‐MS method, including RPLC fraction collection with subsequent CZE‐ESI‐MS analysis .…”

Revealing the potential of capillary electrophoresis/mass spectrometry: the tipping point

“…The sheath‐flow CE/nanoESI‐MS coupling has been successfully used for the analysis of complex protein digest mixtures in a shotgun proteomics strategy, the separation followed by top‐down MS characterization of intact proteins in bacteria secretome, and the characterization of protein glycosylation . The results described demonstrate in a general manner an increase in MS signal intensity by 50‐ to 100‐fold compared with conventional sheath‐liquid CE/MS which enabled the validation of the concept of reducing the volumes of sheath liquid involved in order to enhance CE/MS sensitivity.…”

Glycosylation of biosimilars: Recent advances in analytical characterization and clinical implications

“…However, this represents the overall glycan occupancy and does not indicate the occupancy tailored to distinct glycoforms. In a similar approach with CZE-ESI-MS, site-specific glycan profiling was achieved withiñ 9 min with superior detection limits (i.e., 2 orders of magnitude) and a 200-fold smaller injection volume compared to nano-LC [142]. However, no information on site-occupancy was achieved, and only Fc glycosylation was studied.…”

“…Another interesting approach is the use of CZE-ESI-MS for the relative quantitation of N-glycan species on a site-specific glycopeptide level. Similar to the previously mentioned LC-MS/MS approaches, the proposed CZE based method is not restricted to the analysis of glycopeptides but allows complete primary sequence and quantification of multiple PTMs in a single analytical workflow [142,147]. The use of CZE provides very high separation efficiencies, owing to the limited peak broadening effect in absence of a stationary phase.…”