A Rapid Array-Based Approach to N-Glycan Profiling of Cultured Cells

Abstract

Typically, N-glycosylation studies done on cultured cells require up to millions of cells followed by lengthy preparation to release, isolate, and profile N-glycans. To overcome these limitations, we report a rapid array-based workflow for profiling N-glycan signatures from cells, adapted from imaging mass spectrometry used for on-tissue N-glycan profiling. Using this approach, N-glycan profiles from a low-density array of eight cell chambers could be reported within 4 h of completing cell culture. Approaches are demonstrated that account for background N-glycans due to serum media. Normalization procedures are shown. The method is robust and reproducible, requiring as few as 3000 cells per replicate with a 3–20% coefficient of variation to capture label-free profiles of N-glycans. Quantification by stable isotopic labeling of N-glycans in cell culture is demonstrated and adds no additional time to preparation. Utility of the method is demonstrated by measurement of N-glycan turnover rates due to induction of oxidative stress in human primary aortic endothelial cells. The developed method and ancillary tools serve as a foundational launching point for rapid profiling of N-glycans ranging from high-density arrays down to single cells in culture.

Rapid N-Glycan Profiling of Serum and Plasma by a Novel Slide-Based Imaging Mass Spectrometry Workflow

“…In this study, a comparatively rapid and reproducible slide-based method for N-glycan profiling analysis of serum and plasma N-glycan profiling was presented. The approach possesses the advantages of related glycan profiling protocols for tissues, cells, and antibody capture arrays that our collective group have previously reported, − in that it requires minimal sample processing and no derivatization or purification of PNGase F released glycans. Using only 1–2 μL of serum or plasma, the sensitivity of this method allows for detection of a broad set of N-glycans, comparable to other N-glycan profiling methods, as well as single day analysis timelines.…”

“…Our collective group has developed methods for analyzing released N-glycans from tissues, cells, and immuno-captured serum glycoproteins by MALDI imaging mass spectrometry (MALDI-IMS). − The key to each of these methods is the spraying of a molecular coating of peptide N-glycosidase F (PNGase F) onto a slide surface containing the biological sample, followed by the detection of released N-glycans by MALDI-IMS. We hypothesized that we could adapt techniques from these workflows to create a rapid and sensitive serum N-glycan profiling method.…”

The role of neuraminidase in TLR4‐MAPK signalling and the release of cytokines by lupus serum‐stimulated mesangial cells

“…Sialylated glycoprotein N‐glycans were measured in intact cells that were grown on glass slides and cultured with lupus serum in the absence (six replicate wells) or presence of OP (three replicate wells). Cells were incubated with OP for 16 h and media replaced with 10% lupus serum for 3 h. Following stimulation, cells were prepared for N‐glycan profiling as described previously 38 . Briefly, the array was washed in cold PBS and fixed in neutral buffered formalin followed by delipidation with three replicates of Carnoy's Solution (10% glacial acetic acid, 30% chloroform and 60% 200‐proof ethanol) for three minutes.…”

Applications and continued evolution of glycan imaging mass spectrometry

“…A later orthogonal analysis confirmed that the majority of the detected N‐glycans with at least one fucose residue in the HCC TMA, especially those associated with poor survival, are core‐fucosylated (West et al, 2020). N‐glycan imaging of hepatoblastoma (HepG2) cells in 2019 detected afucosylated, bi‐ and tri‐antennary structures as well as the high mannose species Hex9HexNAc2, suggesting disparate glycoprofiles between liver cancers with different origins (Angel et al, 2019). Antibody panel‐based (APB) N‐glycan imaging, described in a later section, was initially developed with screening for liver malignancies in mind (Black et al, 2019b).…”