Characterisation of a new online nanoLC-CZE-MS platform and application for the glycosylation profiling of alpha-1-acid glycoprotein.

ABSTRACT: The ever-increasing complexity of biological samples to be analysed by mass spectrometry has led to the necessity of sophisticated separation techniques, including multidimensional separation. Despite a high degree of orthogonality, the coupling of liquid chromatography (LC) and capillary zone electrophoresis (CZE) has not gained notable attention in research. Here, we present a heart-cut nanoLC-CZE-ESI-MS platform to analyse intact proteins. NanoLC and CZE-MS are coupled using a four-port valve with an internal nanoliter loop. NanoLC and CZE-MS conditions were optimised independently to find ideal conditions for the combined setup. The valve setup enables an ideal transfer efficiency between the dimensions while maintaining good separation conditions in both dimensions. Due to the higher loadability, the nanoLC-CZE-MS setup exhibits a 280-fold increased concentration sensitivity compared to CZE-MS. The platform was used to characterise intact human alpha-1-acid glycoprotein (AGP), an extremely heterogeneous N-glycosylated protein. With the nanoLC-CZE-MS approach, 368 glycoforms can be assigned at a concentration of 50 μg/mL as opposed to the assignment of only 186 glycoforms from 1 mg/mL by CZE-MS. Additionally, we demonstrate that glycosylation profiling is accessible for dried blood spot analysis (25 μg/mL AGP spiked), indicating the general applicability of our setup to biological matrices. The combination of high sensitivity and orthogonal selectivity in both dimensions makes the here-presented nanoLC-CZE-MS approach capable of detailed characterisation of intact proteins and their proteoforms from complex biological samples and in physiologically relevant concentrations.

SUBMITTER: Stolz A 

PROVIDER: S-EPMC8791864 | biostudies-literature |

REPOSITORIES: biostudies-literature