Mass spectrometric profiling of N-linked oligosaccharides and uncommon glycoform in mouse serum with head and neck tumor

Abstract

N-linked oligosaccharides obtained from total serum of mice with implanted head and neck tumors were analyzed and compared with those from control samples of healthy mice. Methods used include a combination of a derivatization procedure with phenylhydrazine (PHN) and analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Oligosaccharides were enzymatically released from total serum with PNGaseF and purified by high-performance liquid chromatography (HPLC) on a reversed-phase column. Mass spectra contained ion peaks of labeled oligosaccharides and MS/MS experiments provided useful data for the structural elucidation of these compounds. More than 40 N-glycans with compositions characteristic of high-mannose, hybrid, complex, neutral, and sialylated structures were identified in the serum of tumoral mice. Significant differences between samples were observed with respect to the abundances of high mannose and hybrid glycans. These oligosaccharides showed higher relative intensities in the spectra obtained from the cancer sera. Complex sialylated oligosaccharides had similar abundances in both types of sera, with the exception of fucosylated biantennary disialylated oligosaccharide, which was mostly detected with lower abundance in control samples. In the MALDI spectra, several minor species corresponded to uncommon carbohydrates. These structures have been investigated in detail by MS/MS. Among these novel glycoforms, a few sialylated oligosaccharides without a free reducing end were identified. Also, glycans with an extra 60 u were observed and likely feature the presence of a 2-acetamido-2-deoxyoctose residue attached on antennae of 3-or 6-linked mannose. . It has been recognized that altered glycosylation is an universal feature of living cells and several studies have demonstrated that the changes in cellular glycosylation profiles can be also related to aggressive cancer cell behavior, such as tumor cell invasion and metastasis [2][3][4][5][6][7][8]. As cancer cells can produce oligosaccharides that are significantly different from those in normal cells, comparative studies between glycoprotein carbohydrates produced by malignant cells and normal cells may provide useful information for diagnosis, prognosis, and immunotherapy of tumors [9].Mass spectrometry (MS) has been used extensively as the most sensitive method for the analysis of carbohydrate molecules [10 -14]. Moreover, modern MS instruments allow the measurement of unseparated mixtures, and make possible the identification of trace amounts of glycans in biological materials [15]. Nonetheless, biochemical analysis of carbohydrates is generally complicated, not only because of their lower concentration relative to with proteins, but also because of the structural diversity of molecules, anomericity, linkage positions, and branching patterns of monosaccharides. Moreover, carboxylic groups cause sialylated oligosaccharides to ionize with less efficiency than neutral oligosaccharides, making MS analysis of glycans more difficu...

High-Mannose Glycans are Elevated during Breast Cancer Progression

“…For example, Man 5 -Man 7 glycans were present in mice implanted with head and neck tumor but not in healthy mice (29). In a different study, Man 5 -Man 8 glycans were statistically elevated in the soluble cytosolic glycoproteins isolated from invasive and noninvasive breast cancer cells compared with those isolated from normal epithelial cells (32).…”

N-Glycomic Changes in Human Breast Carcinoma MCF-7 and T-Lymphoblastoid Cells After Treatment with Herceptin and Herceptin/Lipoplex

“…The high incidence of oligomannose structures was shown to be associated with human ovarian marker and human follicular lymphoma cells 48,49 and increased levels of high-mannose oligosaccharides were as well observed in sera of animals affected with cancer. 26,31 The peaks corresponding to compositions of hybrid, complex and tri-or tetranantennary sialylated oligosaccharides including their fucosylated analogs, were detected in both cell lines (Figures 2a and 3a). However, in contrast to the high-mannose glycans, these structures were present at low abundances <25% in both lines examined.…”

“…[21][22][23][24][25][26][27][28][29][30][31][32][33] However, less attention has been paid to the study of glycosylation during and after treatment. [34][35][36] In this regard, our present work is aimed to MS investigation of N-glycans in human breast MCF-7 carcinoma and T-lymphoblastoid CEM cells vs oligosaccharide profiles obtained from cells treated by ex-vivo assay with Herceptin alone or combined with Lipofectamine (LipA) and plasmid DNA.…”

Reversed-phase separation methods for glycan analysis

“…Notably, reversed-phase chromatography is sometimes used to desalt the sample or to create a rough separation of highly complex samples and not to obtain fully separated glycans [49, 110, 113, 121, 134]. This full separation of the various glycans is often not necessary when MS detection is used, because ions of a specific m / z can be selected for further analysis or to obtain an extracted ion chromatogram.…”