Isomer-specific chromatographic profiling yields highly sensitive and specific potential N-glycan biomarkers for epithelial ovarian cancer

Abstract

Aberrant glycosylation has been observed for decades in essentially all types of cancer, and is now well established as an indicator of carcinogenesis. Mining the glycome for biomarkers, however, requires analytical methods that can rapidly separate, identify, and quantify isomeric glycans. We have developed a rapid-throughput method for chromatographic glycan profiling using microfluidic chip-based nanoflow liquid chromatography (nano-LC)/mass spectrometry. To demonstrate the utility of this method, we analyzed and compared serum samples from epithelial ovarian cancer cases (n = 46) and healthy control individuals (n = 48). Over 250 N-linked glycan compound peaks with over 100 distinct N-linked glycan compositions were identified. Statistical testing identified 26 potential glycan biomarkers based on both compositional and structure-specific analyses. Using these results, an optimized model was created incorporating the combined abundances of seven potential glycan biomarkers. The receiver operating characteristic (ROC) curve of this optimized model had an area under the curve (AUC) of 0.96, indicating robust discrimination between cancer cases and healthy controls. Rapid-throughput chromatographic glycan profiling was found to be an effective platform for structure-specific biomarker discovery.

Introduction

Glycosylation is an important determinant of protein function, yet it is highly sensitive to its biochemical environment. Major biological changes such as cancer have been repeatedly associated with aberrant glycosylation in humans [1], [2]. These alterations in turn modulate many cancer-related processes, including apoptosis [3], [4], angiogenesis [5], [6], growth factor receptor binding [7], [8], integrin–cadherin function [9], [10], etc. The glycome, thus, serves as a rich source of potential biomarkers for cancer and other diseases [11].

Global profiling of human serum glycans has already identified potential biomarkers for several types of cancer [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24]. However, many of these studies focus on compositional glycan profiling. In contrast, structure-specific glycan profiling has the potential to uncover more robust glycan biomarkers with higher specificity than compositional profiling alone. For example, changes in the glycosidic linkages of single monosaccharide residues have been associated with Alzheimer's disease and pancreatic cancer [25], [26]. Additionally, since each glycan composition can comprise multiple glycan structures, structure-specific glycan profiling provides a significantly larger set of potential biomarkers [27], [28].

In order to gain structure-specific information about the glycome, analytical methods for isomer differentiation and characterization must be applied. Tandem mass spectrometry has historically been used to differentiate between certain targeted glycan linkages [23], [29], [30]; however, this typically requires extra derivatization steps (commonly, permethylation) to render glycans amenable to analysis and furthermore provides only partial information about possible isomers. More recently, ion mobility spectroscopy has been used to attain partial separation of glycan isomers [22], [31]. Chromatographic separation, however, has been the most universally successful method of isomer-specific glycan profiling to date [19], [20], [21], [32], [33], [34].

Chromatographic glycan profiling utilizes isomer-sensitive stationary phases to chromatographically separate complex glycan mixtures. In contrast to tandem MS-centric methods, chromatographic glycan profiling can be performed on native glycans with minimal sample processing [19], [35]. In particular, MS/MS structural elucidation as well as linkage-specific glycosidase digestions have previously shown chip-based porous graphitized carbon (PGC) nano-LC to be highly effective at separating isomeric oligosaccharides, glycans, and glycopeptides with high chromatographic resolution and retention time reproducibility [36], [37], [38], [39], [40]. LC provides a second dimension of separation that complements our previously-developed strategy for compositional glycan profiling by high-resolution MS. By coupling together reproducible isomer-sensitive LC with accurate mass MS, glycan isomers can be separated and rapidly identified according to both retention time and exact mass [33], [34], [36], [37], [38], [41], [42].

Chip-based nano-LC/MS has proven extremely effective for the global separation of serum glycans [19], [43]. Nano-LC/MS offers significantly improved sensitivity over conventional LC/MS or MALDI-MS [16], [17]. In addition, nano-ESI generally produces lower energy ions and therefore yields less in-source fragmentation than MALDI. Integration of these features within a microfluidic chip vastly simplifies analysis while providing unparalleled retention time reproducibility [19], [44]. Coupling chip-based nano-LC with a time-of-flight (TOF) MS detector imparts the added benefits of high mass accuracy and dynamic range of detection [45], [46], [47], [48].

Some groups have previously reported that porous graphitized carbon can separate not only glycan isomers, but also alpha and beta anomers [34]. One common sample preparation technique is to chemically reduce native N-glycans, removing the possibility of anomerization and simplifying analysis of the resulting chromatogram [33], [34], [41], [43]. However, the high chromatographic resolution and retention time reproducibility exhibited by the chip-based nano-LC format enable a technological rather than chemical solution to this problem – using a combination of accurate mass and retention time, native glycan isomer peaks, including anomers as well as regioisomers, may be easily compared and quantified across many chromatographic runs [19]. Additionally, analysis of native glycans sidesteps the additional sample handling, extended sample cleanup, and inevitable chemical artifacts associated with chemical derivatization strategies. As a result, native glycans can be isolated and analyzed with less sample processing and greater quantitative precision than reduced or otherwise-derivatized glycans, making them the ideal analyte for biomarker applications.

We have developed a rapid-throughput method for comprehensive, isomer-specific chromatographic profiling of native serum glycans and applied it to glycan biomarker discovery. Chip-based porous graphitized carbon nano-LC/MS was used to quickly separate and quantify native, underivatized N-glycans. Serum samples from epithelial ovarian cancer cases and healthy control individuals were profiled and compared both by overall compositional abundance and in relation to specific isomers. Statistical tests were performed to detect significant differences in cancer cases vs. healthy controls as well as determine the discriminatory power of potential glycan biomarkers. Rapid-throughput chromatographic glycan profiling was shown to be a powerful platform for glycan biomarker discovery, providing rapid yet detailed characterization and quantitation of large sample sets.