N-linked Glycosylation Profiling of Pancreatic Cancer Serum Using Capillary Liquid Phase Separation Coupled with Mass Spectrometric Analysis

Abstract

Glycoproteins play important roles in various biological processes including intracellular transport, cell recognition, and cell-cell interactions. The change of the cellular glycosylation profile may have profound effects on cellular homeostasis and malignancy. Therefore, we have developed a sensitive screening approach for the comprehensive analysis of N-glycans and glycosylation sites on human serum proteins. Using this approach, N-linked glycopeptides were extracted by double lectin affinity chromatography. The glycans were enzymatically cleaved from the peptides and then profiled using capillary hydrophilic interaction liquid chromatography coupled online with ESI-TOF MS. The structures of the separated glycans were determined by MALDI quadrupole ion-trap TOF mass spectrometry in both positive and negative modes. The glycosylation sites were elucidated by sequencing of PNGase F modified glycopeptides using nanoRP-LC-ESI-MS/MS. Alterations of glycosylation were analyzed by comparing oligosaccharide expression of serum glycoproteins at different disease stages. The efficiency of this method was demonstrated by the analysis of pancreatic cancer serum compared to normal serum. Ninety-two individual glycosylation sites and 202 glycan peaks with 105 unique carbohydrate structures were identified from approximately 25 mug glycopeptides. Forty-four oligosaccharides were found to be distinct in the pancreatic cancer serum. Increased branching of N-linked oligosaccharides and increased fucosylation and sialylation were observed in samples from patients with pancreatic cancer. The methodology described in this study may elucidate novel, cancer-specific oligosaccharides and glycosylation sites, some of which may have utility as useful biomarkers of cancer.

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

“…[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.…”

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

“…Serum of patients with cirrhosis showed a heterogeneous distribution of N-glycans compared with healthy samples [23]. In a study on human pancreatic cancer, serum samples contained N-glycans with increased fucosylation and sialylation relative to those from healthy individuals [24]. Samples in the study of ovarian cancer patients also showed a number of neutral oligosaccharides that appeared related to cancer [25].…”

Label-free quantitation: A new glycoproteomics approach

“…When a mixture of glycopeptides is analyzed, one current roadblock is being able to determine why the particular glycopeptide ion has changed in abundance, i.e., is it due to changes in glycosylation on a given protein or due to changes in the protein's concentration, relative to the other species being analyzed [4]? Our method, which characterizes the entire glycosylation profile for a given glycoprotein as part of the quantitation process, is ideally suited to solving this problem.…”

“…This introduces many problems in biomarker discovery, such as not being able to identify whether or not the glycan's concentration increased because a protein containing that glycan was overexpressed, or if one or several proteins' glycosylation profile changed, causing the glycan to be more abundant, even though the protein level(s) are not altered [4]. In contrast to glycan analysis, glycopeptide analysis provides glycosylation site-specific information for purified proteins [17,20,21], and it could potentially be useful in distinguishing between glycosylation changes and protein expression changes, since the protein information is encoded in the glycopeptide.…”

“…These methods have the potential to compare multiple samples with ease. Changes in intensities of mass spectral peaks have been assessed by comparing different sample sets through either glycan analysis [4] or glycopeptide analysis [32]. Because signal intensities can vary between mass spectrometric samples, label-free approaches are not as commonly used for quantitative analysis [26,33].…”