Conventional protocols for cellular N-glycan profiling often need several time-consuming and labor-intensive steps, and a large amount of material (5–20 × 106 cells) is required. In this study, an optimized co-derivatization method was applied for convenient, rapid and highly-sensitive analysis of cellular N-glycan. Taking human cervical carcinoma cells (HeLa) as a model, the required amount for comprehensive cellular N-glycans analysis was reduced down to an original cell number of 105 and the total analysis time was decreased from several days to several hours. Good reproducibility of the method was also obtained with the CV averaging to less than 13.1%. In addition, compared to permethylation derivatization, more than 5-fold sensitivity improvement was achieved and more concise and clear mass spectra were obtained with the new developed method. As a preliminary study, this method was also successfully applied to reveal the difference in cellular N-glycan profiling of two heterogeneous live cell lines, human normal liver cells (L-02) and human hepatocyte carcinoma cells (HepG2), showing great potential for high-throughput analysis of N-glycans from limited cell samples, such as primary cell lines, cells obtained from cell sorting and small size cancer specimens.

•Instable lactones were converted to more stable N-methyl amides.•Byproducts and side reaction was minimized in our method.•Our method is beneficial to the quantification of sialylated glycans.Sialic acids usually locate at the terminal of many glycan structures in either α(2,3) or α(2,6) linkage, playing different roles in various biological and pathological processes. Several linkage specific carboxyl derivatization methods have been reported to discriminate between α(2,3) and α(2,6)-linked sialic acids by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Among them, ethyl esterification was recently reported to achieve linkage specific derivatization between α(2,3) and α(2,6)-linked sialic acids with good selectivity. However, the method suffered from the instability of the generated lactones and byproducts of the derivatives. To overcome these shortcomings, a solid-phase two step derivatization method was introduced to convert the α(2,6)-linked sialic acid into ethyl esters and the α(2,3)-inked counterparts into N-methyl amides, respectively. Under the optimized derivatization conditions, our method was able to achieve accurate relative quantification of N-glycan as well as their corresponding sialylated linkage types, superior to the ethyl esterification method. The solid phase derivatization strategy was further applied to investigate N-glycans from biosimilar antibody drug and human serum from patients and healthy volunteers. This method has the potential to be used in the biomarker discovery and pharmaceutical industry.

•Glycosylamine could be achieved by rapid deglycosylation within 5 min.•More than 50 fold improvement in sensitivity was obtained by TMPP-Ac-OSu derivatization.•Complex samples could be analyzed in combination with methylamidation.Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become an important technology for glycan analysis due to its ease of operation, short analysis time and impurity tolerance. However, the low ionization efficiency of N-glycans led to the difficulty in analyzing glycans of low abundance in complex biological samples due to the lack of basic site for protonation. Therefore, highly sensitive method for the glycans analysis is in urgent demand. Here we report a new strategy to introduce a permanent charge at the reducing end of N-linked glycans by a one pot reaction, where glycosylamines that were obtained by rapid deglycosylation within 5 min were labeled with N-succinimidyloxycarbonylmethyl tris (2,4,6- trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu). With TMPP-Ac labeling, more than 50 fold enhancement in the sensitivity of method was achieved for neutral glycans from ribonuclease B (RNase B) in comparison to their native counterparts. In combination with methylamidation of sialic acid residues, this novel developed strategy could also be used for sialylated glycans analysis from sialoglycoproteins and complex serum sample. As a result, more than 50 glycans were detected with only 25 nL human serum sample.

•A co-derivatization strategy using TMPP-Ac-OSu and methylamidation in coupling with pronase E digestion.•Highly sensitive glycan profile analysis by MALDI-MS.•Stabilized sialylated oligosaccharides during MALDI-MS analysis.As one of the most prevalent and complex post-translational modifications in biological systems, proteins glycosylation has drawn considerable attention in recent decades. Dissociation of the carbohydrates from glycoproteins may be the prerequisite step of glycomics experiments, which commonly performed by specific proteolysis. In this study, an alternative strategy was reported with nonspecific proteolysis in coupling with co-derivatization of TMPP-Ac and methylamidation for glycan moieties analysis by MALDI-MS. With the co-derivatization, a permanent positive charge was introduced to the Asn-glycans and the carboxylic groups were neutralized by methylamidation simultaneously. As a result, approximately 20 and 50-fold enhancement in the detection sensitivity was achieved for asialo-Asn and disialo-Asn respectively in comparison to their native counterparts. Ultimately, this developed strategy was successfully validated using three model glycoproteins, including ribonuclease B, ovalbumin and transferrin.

The analysis of isomeric glycans is a challenging task. In this work, a new strategy was developed for isomer-specific glycan profiling using nanoLC-MS with PGC as the stationary phase. Native glycans were derivatized in the presence of methylamine and trispyrrolidinophosphonium hexafluorophosphate and reduced by the ammonia–borane complex. Methylamidation stabilized the retention time and peak width and improved the detection sensitivity of sialylated glycans to 2–80-fold in comparison to previous ESI-MS methods using the positive-ion mode. Up to 19 tetrasialylated glycan species were identified in the derivatized human serum sample, which were difficult to detect in the sample without derivatization. Furthermore, due to high detection sensitivity and chromatographic resolution, more isomeric glycans could be identified from the model glycoprotein Fetuin and the human serum sample. As a result, up to seven isomers were observed for the disialylated biantennary glycan released from Fetuin, and three of them were identified for the first time in this study. Using the developed analytical strategy, a total of 293 glycan species were obtained from the human serum sample, representing an increase of over 100 peaks in comparison to the underivatized sample. The strategy greatly facilitates the profiling of isomeric glycans and the analysis of trace-level samples.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been a major approach for glycan analysis. However, the preferential cleavage of the sialic acid moiety by in- and post-source decay influences the determination of sialylated glycans by MALDI-MS. Many chemical derivatization methods were introduced to stabilize the sialylated glycan during MALDI-MS. Among current derivatization methods, methylamidation is a promising means for simultaneous analysis of natural sialylated glycans regardless of their sialic acid linkage types. Here, a novel derivatization method was developed, in which proteins were conjugated on the solid-phase support in order to stabilize the sialic acids by methylamidation and to reduce sample loss and contamination during the derivatization process. This derivatization strategy was used to investigate N-glycans from fetuin, a glycoprotein containing different types of complex N-glycans. The developed method was also applied to the N-glycan profiling of human serum from patients and healthy volunteers. Results were consistent with N-glycan profiling by HPLC-fluorescence detection. This new method provided a sensitive, simple, and robust approach for profiling glycan structures of complex samples.