Sialic acids decorate the surface of glycoproteins and play important roles in a variety of pathological processes. Although the mass spectrometry (MS) based linkage-specific analysis of sialylated N-glycopeptide is developing rapidly, quantitative analysis of these isomers still remains a challenge. Herein, we reported a novel quantitative strategy that can unambiguously identify and relatively quantify linkage-specific N-glycopeptides using ion mobility mass spectrometry (IM-MS). Without the assistance of derivatization, this method can relatively quantify sialic acid isomers of intact glycopeptides by using their characteristic fragment ions in IM-MS. Moreover, good linearity (R 2 > 0.99) of relative quantification within a dynamic range of 2 orders of magnitude and high reproducibility (coefficient of variation (CV) < 10%, n = 3) were demonstrated. Finally, our results illustrated the aberrant sialylation of haptoglobin (Hp) in hepatocellular carcinoma (HCC), where the ratios of α2,3 to α2,6 sialylation of seven N-glycopeptides were found to be significantly altered (p < 0.01) in HCC individuals (n = 27) compared with healthy controls (n = 27).

Sialylated N-glycan isomers with α-2,3 and α-2,6 linkages play crucial and distinctive roles in diverse physiological and pathological processes. Changes of α-2,3-linked sialic acids in sialylated N-glycans are especially important in monitoring the initiation and progression of diseases. However, the specific analysis of α-2,3-sialylated N-glycan linkage isomers remains challenging due to their extremely low abundance and technical limitations in separation and detection. Herein, we designed an integrated strategy that combines linkage-specific derivatization and a charge-sensitive separation method based on microfluidic chip capillary electrophoresis−mass spectrometry (microchip CE−MS) for specific analysis of α-2,3-sialylated N-glycan linkage isomers for the first time. The α-2,6and α-2,3-sialic acids were selectively labeled with methylamine (MA) and N,Ndimethylethylenediamine (DMEN), respectively, which selectively makes α-2,3-sialylated N-glycans positively charged and realizes online purification, concentration, and discrimination of α-2,3-sialylated N-glycans from other N-glycans in microchip CE−MS. This new approach was demonstrated with standard multisialylated N-glycans, and it was found that only the α-2,3-sialylated N-glycans migrated and were detected in order according to the number of α-2,3-sialic acids. Finally, this strategy was successfully applied in highly sensitive profiling and reproducible quantitation of the serum α-2,3-sialylated N-glycome from ovarian cancer (OC) patients, where 7 of 33 detected α-2,3-sialylated N-glycans significantly changed in the OC group compared with healthy controls.

Kaixin Powder (KXP) is a classic formula for treating morbid forgetfulness in ancient China. To guarantee the efficacy and safety of KXP, a simple and accurate HPLC–DAD method has been established and validated for the quantitative analysis of seven bioactive compounds in KXP. Dehydrotumulosic acid (DTU) and dehydrotrametenolic acid (DTR) were quantified in KXP for the first time. Good chromatographic separation was conducted on a Kromasil 100-5 C 18 column (250 mm × 4.6 mm, 5 μm) by gradient elution using mobile phases containing acetonitrile and 0.1% formic acid aqueous solution at different detection wavelengths. The calibration curves of each compound showed good linearity ( r ≥ 0.9990), and the LOD and LOQ were in the ranges of 0.01–0.10 and 0.03–0.40 μg/mL, respectively. The relative standard deviations (RSDs) of intra-day and inter-day precisions were in the ranges of 0.45–1.74% and 0.56–2.32%, respectively. All recoveries were in the range of 93.6–105.5% with an RSD no more than 2.77%. These quantification results of seven compounds determined in the samples were further confirmed by HPLC–QTOF-MS/MS. This study provides a useful and simple method for analyzing the major bioactive compounds and improves the quality assessment research of KXP.

ObjectiveFor locally advanced cervical cancer (LACC), patients who respond to chemotherapy have a potential survival advantage compared to nonresponsive patients. Thus, it is necessary to explore specific biological markers for the efficacy of chemotherapy, which is beneficial to personalized treatment.MethodsIn the present study, we performed a comprehensive screening of site-specific N-glycopeptides in serum glycoproteins to identify glycopeptide markers for predicting the efficacy of chemotherapy, which is beneficial to personalized treatment. In total, 20 serum samples before and after neoadjuvant chemotherapy (NACT) from 10 LACC patients (NACT response, n=6) and NACT nonresponse, n=4) cases) were analyzed using LC-MS/MS, and 20 sets of mass spectrometry (MS) data were collected using liquid chromatography coupled with high-energy collisional dissociation tandem MS (LC-HCD-MS/MS) for quantitative analysis on the novel software platform, Byos. We also identified differential glycopeptides before and after chemotherapy in chemo-sensitive and chemo-resistant patients.ResultsIn the present study, a total of 148 glycoproteins, 496 glycosylation sites and 2279 complete glycopeptides were identified in serum samples of LACC patients. Before and after chemotherapy, there were 13 differentially expressed glycoproteins, 654 differentially expressed glycopeptides and 93 differentially expressed glycosites in the NACT responsive group, whereas there were 18 differentially expressed glycoproteins, 569 differentially expressed glycopeptides and 99 differentially expressed glycosites in the NACT nonresponsive group. After quantitative analysis, 6 of 570 glycopeptides were identified as biomarkers for predicting the sensitivity of neoadjuvant chemotherapy in LACC. The corresponding glycopeptides included MASP1, LUM, ATRN, CO8A, CO8B and CO6. The relative abundances of the six glycopeptides, including MASP1, LUM, ATRN, CO8A, CO8B and CO6, were significantly higher in the NACT-responsive group and were significantly decreased after chemotherapy. High levels of these six glycopeptides may indicate that chemotherapy is effective. Thus, these glycopeptides are expected to serve as biomarkers for predicting the efficacy of neoadjuvant chemotherapy in locally advanced cervical cancer.ConclusionThe present study revealed that the N-glycopeptide of MASP1, LUM, ATRN, CO8A, CO8B and CO6 may be potential biomarkers for predicting the efficacy of chemotherapy for cervical cancer.

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