The Analysis of Human Serum N-Glycosylation in Patients with Primary and Metastatic Brain Tumors

Abstract

The identification of patients with different brain tumors is solely built on imaging diagnostics, indicating the need for novel methods to facilitate disease recognition. Glycosylation is a chemical modification of proteins, reportedly altered in several inflammatory and malignant diseases, providing a potential alternative route for disease detection. In this paper, we report the quantitative analysis of serum N-glycosylation of patients diagnosed with primary and metastatic brain tumors. PNGase-F-digested and procainamide-labeled serum glycans were purified by magnetic nanoparticles, followed by quantitative liquid chromatographic analysis. The glycan structures were identified by the combination of single quad mass spectrometric detection and exoglycosidase digestions. Linear discriminant analysis provided a clear separation of different disease groups and healthy controls based on their N-glycome pattern. Altered distribution of biantennary neutral, sialylated but nonfucosylated, and sialylated–fucosylated structures were found to be the most significant changes. Our results demonstrate that serum glycosylation monitoring could improve the detection of malignancy.

High-Throughput Glycomic Methods

“…27,69,118,119 Although it results in high fluorescent signals, it is not easily ionizable, hindering MS characterization of labeled N-glycan species. Several other labels have recently been more and more applied, e.g., procainamide (ProA), 120 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate (AQC), 121 and RapiFluor-MS 122 (Figure 7), showing equivalent or enhanced fluorescence compared to 2-AB, as well as ionization in the cases of ProA and RapiFluor-MS due to the introduction of a charged tertiary amine tag. 117,120,123,124 Moreover, INLIGHT labeling strategy has been used to increase hydrophobicity and ionization of Nglycans for more efficient RP-LC-MS analysis.…”

“…Several other labels have recently been more and more applied, e.g., procainamide (ProA), 120 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate (AQC), 121 and RapiFluor-MS 122 (Figure 7), showing equivalent or enhanced fluorescence compared to 2-AB, as well as ionization in the cases of ProA and RapiFluor-MS due to the introduction of a charged tertiary amine tag. 117,120,123,124 Moreover, INLIGHT labeling strategy has been used to increase hydrophobicity and ionization of Nglycans for more efficient RP-LC-MS analysis. 125,126 A typical fluorescent labeling reaction with 2-AB or ProA is performed at 65 °C for 2−3 h, creating a balance between labeling efficiency and the loss of sialic acids.…”

“…Additionally, magnetic nanoparticles can also be used as a cleanup approach. 120,131 Different variations of the standard glycan preparation workflows have recently become available in a kit format, e.g., GlycoWorks RapiFluor-MS N-Glycan Kit (Waters), Advance-Bio Gly-X N-Glycan Prep Kits (Agilent), and Glycoprofile Labeling Kits (Merck), expediting their application in basic HT glycomic research and biomarker discovery as well as automation.…”

“…Of note, while PNGase F is widely used in released N -glycan analysis, it appears to have poor efficiency for releasing highly truncated N -glycans with, e.g., only chitobiose or a single N -acetylglucosamine that appear to be rather common. , Sometimes, an additional step of a cleanup procedure is being used after deglycosylation, facilitating the fluorescent labeling reaction efficiency. , Historically, 2-AB has been the most widely used fluorescent label, starting from the first HT studies performed on the level of thousand samples. ,,, Although it results in high fluorescent signals, it is not easily ionizable, hindering MS characterization of labeled N -glycan species. Several other labels have recently been more and more applied, e.g., procainamide (ProA), 6-aminoquinolyl- N -hydroxysuccinimidyl carbamate (AQC), and RapiFluor-MS (Figure ), showing equivalent or enhanced fluorescence compared to 2-AB, as well as ionization in the cases of ProA and RapiFluor-MS due to the introduction of a charged tertiary amine tag. ,,, Moreover, INLIGHT labeling strategy has been used to increase hydrophobicity and ionization of N -glycans for more efficient RP-LC-MS analysis. , A typical fluorescent labeling reaction with 2-AB or ProA is performed at 65 °C for 2–3 h, creating a balance between labeling efficiency and the loss of sialic acids. With the development of rapid labeling chemistries (Figure ), e.g., AQC, InstantAB, InstantPC, and RapiFluor-MS, the reaction time is significantly decreased to only 5 min, making this approach even more HT.…”

“…The cleanup procedure is generally performed by HILIC-SPE using different hydrophilic stationary phases, e.g., hydrophilic filters (in the form of 96-well plates) , and hydrophilic bead-based cartridges or plates , because these have proven to be effective in the removal of excess reagents and proteins from the previous sample preparation steps. Additionally, magnetic nanoparticles can also be used as a cleanup approach. , …”