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A rapid and sensitive N-glycan profiling strategy for MALDI-MS incorporating the use of deglycosylation with microwave assistance and the co-derivatization of glycosylamine labeling with tris(2,4,6-trimethoxyphenyl)phosphonium acetic acid N-hydroxysuccinimide ester (TMPP-Ac-OSu) and methylamidation has been developed in this work. Notably, highly efficient release and tagging of N-glycans from ribonuclease B was achieved in less than 90 min, providing up to 35-fold enhancement of MALDI-MS sensitivity with comparison to underivatized N-glycans. After further validation with other two standard glycoproteins (ovalbumin and bovine fetuin), the proposed strategy was applied to human serum for preliminary pathological analysis of N-glycans between healthy and lung cancer individuals. As a result, significant differences ( T test p value <0.01) of 6 glycan structures were determined from 54 detected N-glycan structures with only 50 nL of loading amount and further confirmed through PCA and ROC (AUC) analyses between two sample sets. Subsequently, the trend of each lung cancer stage and controls in expression of the selected glycans was implemented with T test and box-plots. Accordingly, these structures can be used as potential lung cancer glycan-based biomarkers and for further definition of cancer progression highlighting the ability of proposed method to rapidly and efficiently analyze N-glycome present in human serum. [ABSTRACT FROM AUTHOR]

Monitoring glycosylation of the mAbs have been emphasized and routinely characterized in biopharmaceutical industries because the carbohydrate components are closely related to the safety, efficacy, and consistency of the antibodies. In this study, the comprehensive glycan profiling of a biosimilar candidate of cetuximab was successfully characterized using Normal phase high-performance liquid chromatography (NP-HPLC) in combination with Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The presence of minor N-linked glycans containing sialic acid lactone residues (NeuAcLac) was observed in the biosimilar for the first time, which could influence the quantitative analysis of sialylated glycans and interfere with quantification of neutral glycans when it was analyzed by high performance liquid chromatography fluorescence (HPLC-FL). To overcome this issue, mild alkali treatment was used to hydrolyze lactone of the sialic acid to their neutral formation, which had no impact on the analysis of other glycans before and after the treatment. As a result, the mild alkali treatment might be helpful to obtain quantitative glycan profiling of the mAbs drugs with enhanced accuracy and robustness. [ABSTRACT FROM AUTHOR]

Profiling of glycans released from proteins is very complex and important. To enhance the detection sensitivity, chemical derivatization is required for the analysis of carbohydrates. Due to the interference of excess reagents, a simple and reliable purification method is usually necessary for the derivatized oligosaccharides. Various SPE based methods have been applied for the clean-up process. To demonstrate the differences among these methods, seven types of self-packed SPE cartridges were systematically compared in this study. The optimized conditions were determined for each type of cartridge and it was found that microcrystalline cellulose was the most appropriate SPE material for the purification of derivatized oligosaccharide. Normal phase HPLC analysis of the derivatized maltoheptaose was realized with a detection limit of 0.12 pmol (S N−1 = 3) and a recovery over 70%. With the optimized SPE method, relative quantification analysis of N-glycans from model glycoproteins were carried out accurately and over 40 N-glycans from human serum samples were determined regardless of the isomers. Due to the high stability and sensitivity, microcrystalline cellulose cartridge showed potential applications in glycomics analysis. [ABSTRACT FROM AUTHOR]

N-glycans that are fluorescently tagged by glycosylamine acylation have become a promising way for glycan biomarker discovery. Here, we describe a simple and rapid method using Fmoc N-hydroxysuccinimide ester (Fmoc-OSu) to label N-glycans by reacting with their corresponding intermediate glycosylamines produced by microwave-assisted deglycosylation. After optimizing reaction conditions, this derivatization reaction can be effectively achieved under 40 °C for 1 h. Moreover, the comparison of fluorescent intensities for Fmoc-OSu, Fmoc-Cl and 2-AA labeling strategies were also performed. Among which, the fluorescent intensities of Fmoc-OSu labeled glycan derivatives were approximately 5 and 13 times higher than that labeled by Fmoc-Cl and 2-AA respectively. Furthermore, the developed derivatization strategy has also been applied for analyzing serum N-glycans, aiming to screen specific biomarkers for early diagnosis of lung squamous cell cancer. More interestingly, the preparation of free reducing N-glycan standards have been achieved by the combination of HPLC fraction of Fmoc labeled glycan derivatives and Fmoc releasing chemistry. Overall, this proposed method has the potential to be used in functional glycomic study. Image 1 • The combination of Fmoc-OSu derivatization with microwave-assisted digestion was developed for N-glycan analysis. • High-sensitivity detection of N-glycan was achieved. • Free reducing N-glycans from glycoproteins have been prepared by releasing Fmoc residue. • Several potential N-glycan biomarkers was identified for early diagnosis of lung squamous cell cancer. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) has become one of the most common cancers worldwide and the fifth most prevalent cancer in China with an upward trend in incidence rates. Altered glycosylation significantly affects the structural and functional changes in immunoglobulin G (IgG) and was consequently associated with disease progression. In this study, we explored the association of glycosylation with CRC prognosis through characterizing serum IgG N -glycans derived from individuals consisting of normal, benign colorectal and CRC cohorts in discovery set. Statistical analysis showed nine of IgG N -glycans were differentially expressed in disease groups compared with controls. Additionally, five out of them were still significantly changed in CRC patients at all tumor node metastasis (TNM) stages as compared with controls. Principal component analysis (PCA) indicated obvious differentiation of benign and cancer patients from normal individuals. Further diagnostic performance of receiver operator curve (ROC) analysis demonstrated at least moderately accurate area under the curve (AUC) score with preferable sensitivity and specificity, suggesting these five IgG N -glycans were probably correlated with CRC progression. Significantly, this result has been verified in validation set. Moreover, IgG N -glycosylation analysis suggested that core-fucosylation, sialylation and sialo core-fucosylation were possibly related to the development of CRC. Significance In-depth IgG N -glycome profiling of colorectal benign patients, colorectal cancer and normal individuals reveals differentially expression levels of N -glycosylation. Differing from serum comprehensive glycomes, profiling of specific serum glycoprotein contributes to more detailed understanding of biological relevance of glycosylation alterations in disease prognosis. Additionally, the high-throughput technique, MADLI-TOF-MS could absolutely relieve manual stress from sample preparation and accelerate information acquisition as compared to another recent analytical method. Moreover, we introduced a fast and easy data processing of MS exported files based on a software solution, which had the advantage of avoiding time consuming in manually searching and calculating the interesting peaks, and automatically producing the average percentage of each glycan over usual operation with Microsoft Excel. Besides, it may be useful for large-scale study. [ABSTRACT FROM AUTHOR]

Glycosylation is one of the most important post-translational modifications of protein. Recently, global profiling of human serum glycomics has become a noninvasive method for cancer-related biomarker discovery and many studies have focused on compositional glycan profiling. In contrast, structure-specific glycan profiling may provide more potential biomarkers with higher specificity than compositional profiling. In this work, N-glycans released from human serum were neutralized with methylamine and reduced by ammonia-borane complex prior to profiling using nanoLC-ESI-MS with porous graphitized carbon (PGC) and relative abundances of over 280 isomers were compared between pancreatic cancer (PC) cases (n = 32) and healthy controls (n = 32). Statistical analysis identified 25 specific-isomeric biomarkers with significant differences (p-value < 0.05). ROC and PCA analysis were performed to assess the potential biomarkers which were identified as being significantly altered in cancer. The AUCs of the significantly changed specific-isomers were ranging from 0.712 to 0.949. In addition, with the combination of all potential biomarkers, a higher AUC of 0.976 with sensitivity (93.5%) and specificity (90.6%) was obtained. Overall, the proposed strategy coupled to relative quantitative analysis of isomeric glycans make it possible to discover new biomarkers for the diagnosis of PC. Significance Pancreatic cancer (PC) has a poor prognosis with a five-year survival rate <5%. Therefore, a strategy for accurate diagnosis of PC is indeed required. In this paper, a dual-derivatized strategy for structure-specific glycan profiling has been used and according to our best knowledge, this is the first application of this strategy for PC biomarker discovery, in which the separation, identification and relative quantification of isomeric glycans can be simultaneously obtained. In addition, by in-depth analysis of isomeric glycans, the full description of the stereo- and region- diversity of glycans can also be achieved, which might provide more potential information for PC biomarker discovery. [ABSTRACT FROM AUTHOR]

Quantitative analysis of glycans is an emerging field in glycomic research. Herein we present a rapid and effective dual-labeling strategy, in the combination of isotopic derivatization of N -glycosylamine-based glycans by d0/d5-benzoyl chloride and methylamidation of sialic acids, to relatively quantify both neutral and sialylated N -glycans simultaneously by MALDI-MS. The derivatization efficiencies were increased by microwave-accelerated deglycosylation which not only largely reduce the time of glycoprotein deglycosylation but also inhibit the hydrolysis of intermediate glycosylamines produced by PNGase F digestion. Three model glycoproteins, including RNase B, bovine fetuin and IgG from human serum, were applied to validate this technique. Results showed that the glycans from microgram level of glycoprotein can be successfully quantified with high reproducibility and the whole time of analytical procedure was shortened to 4 h. Furthermore, this proposed method was applied for the comparative analysis of N -glycans from serum of healthy donors and multiple myeloma patients. It was found that five N -glycans may be as the potential biomarkers for rapid detection of early multiple myeloma, indicating the feasibility of this strategy in monitoring subtle quantitative differences of serum glycomics. [ABSTRACT FROM AUTHOR]

Robust, efficient identification and accurate quantification of N -glycans are of great significance in N -glycomics analysis. Here, a simple and rapid derivatization method, based on the combination of microwave-assisted deglycosylation and 6-aminoquinolyl- N -hydroxysuccinimidyl carbamate (AQC) labeling, was developed for the analysis of N -glycan by high performance liquid chromatography with fluorescence detection (HPLC-FLD). After optimizing various parameters affecting deglycosylation and derivatization by RNase B, the time for N -glycan labeling was shortened to 50 min with ∼10-fold enhancement in detection sensitivity comparing to conventional 2-aminobenzoic acid (2-AA) labeling method. Additionally, the method showed good linearity (correlation coefficients > 0.991) and reproducibility (RSD < 8.7%). These advantages of the proposed method were further validated by the analysis of complex samples, including fetuin and human serum. Investigation of serum N -glycome for preliminary diagnosis of human lung cancer was conducted, where significant changes of several N -glycans corresponding to core-fucosylated, mono- and disialylated glycans have been evidenced by a series of statistical analysis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Chemical derivatization of glycans is a common strategy to increase the analytical performance of MALDI-MS-based glycan profiling techniques. Hydrazide, one of the most popular tags, offers important advantages including allowing purification-free procedures. Several hydrazides have thus been used for glycomics combined with an on-target strategy to further simplify the analytical procedures. Usually, gentle heating and mildly acidic conditions with somewhat long reaction times are needed for these hydrazide derivatizations to reach a high reaction efficiency, which makes the current hydrazide tags not yet perfectly conducive to high-throughput analysis. To further optimize these hydrazide tags for high-throughput analysis, based on the structure of a reported hydrazide and the theoretical calculations, a new cationic hydrazide tag, 4-(hydrazinecarbonyl)- N , N , N -trimethylbenzenaminium (HTMBA), was designed, synthesized and tested in this work. HTMBA could completely derivatize glycans at room temperature in several seconds under very mildly acidic conditions (<3% acetic acid). A 19-fold enhancement in the signal intensity was obtained without interference from alkali adduct ions in the MALDI-MS detection of HTMBA-labeled maltoheptaose. To broaden the applicability of HTMBA, an HTMBA on-target derivatization (HOD) strategy was developed and fully validated with maltoheptaose and RNase B, and the method showed a good repeatability and stability. Finally, the HOD strategy was successfully applied to serum samples, 44 glycans in human serum were detected, and the O-acetylation information of sialic acid in horse serum was preserved. These results showed that the HOD strategy was suitable for the MS-based rapid analysis of all glycoforms in complex biological samples. Image 1 • Theoretical calculations assisted design of a new cationic hydrazide tag HTMBA. • HTMBA derivatization enhances 19-fold the signal intensity of glycans in MALDI-MS. • On-target derivatization of glycans at room temperature was achieved with HTMBA. • 44 glycans of human serum were detected in MALDI-MS with HOD strategy. • O-acetylated glycans of horse serum were detected in MALDI-MS with HOD strategy. [ABSTRACT FROM AUTHOR]