Changes in the levels and compositions of Nglycans released from serum and plasma glycoproteins have been assessed in many diseases across many large clinical sample cohorts. Assays used for N-glycan profiling in these fluids currently require multiple processing steps and have limited throughput, thus diminishing their potential for use as standard clinical diagnostic assays. A novel slide-based N-glycan profiling method was evaluated for sensitivity and reproducibility using a pooled serum standard. Serum was spotted on to an amine-reactive slide, delipidated and desalted with a series of washes, sprayed with peptide N-glycosidase F and matrix, and analyzed by MALDI-FTICR or MALDI-Q-TOF mass spectrometry. Routinely, over 75 N-glycan species can be detected from one microliter of serum in less than 6.5 h. Additionally, endoglycosidase F3 was applied to this workflow to identify core-fucosylated N-glycans and displayed the adaptability of this method for the determination of structural information. This method was applied to a small pooled serum set from either obese or nonobese patients that had breast cancer or a benign lesion. This study confirms the reproducibility, sensitivity, and adaptability of a novel method for N-glycan profiling of serum and plasma for potential application to clinical diagnostics.

Expressed prostatic secretions (EPS), also called post digital rectal exam urines, are proximal fluids of the prostate that are widely used for diagnostic and prognostic assays for prostate cancer. These fluids contain an abundant number of glycoproteins and extracellular vesicles secreted by the prostate gland, and the ability to detect changes in their N-glycans composition as a reflection of disease state represents potential new biomarker candidates. Methods to characterize these N-glycan constituents directly from clinical samples in a timely manner and with minimal sample processing requirements are not currently available. In this report, an approach is described to directly profile the N-glycan constituents of EPS urine samples, prostatic fluids and urine using imaging mass spectrometry for detection. An amine reactive slide is used to immobilize glycoproteins from a few microliters of spotted samples, followed by peptide N-glycosidase digestion. Over 100 N-glycan compositions can be detected with this method, and it works with urine, urine EPS, prostatic fluids, and urine EPS-derived extracellular vesicles. A comparison of the N-glycans detected from the fluids with tissue N-glycans from prostate cancer tissues was done, indicating a subset of N-glycans present in fluids derived from the gland lumens. The developed N-glycan profiling is amenable to analysis of larger clinical cohorts and adaptable to other biofluids.

Sialic acid isomers attached in either α2,3 or α2,6 linkage to glycan termini confer distinct chemical, biological, and pathological properties, but they cannot be distinguished by mass differences in traditional mass spectrometry experiments. Multiple derivatization strategies have been developed to stabilize and facilitate the analysis of sialic acid isomers and their glycoconjugate carriers by high-performance liquid chromatography, capillary electrophoresis, and mass spectrometry workflows. Herein, a set of novel derivatization schemes are described that result in the introduction of bioorthogonal click chemistry alkyne or azide groups into α2,3and α2,8-linked sialic acids. These chemical modifications were validated and structurally characterized using model isomeric sialic acid conjugates and model protein carriers. Use of an alkyne-amine, propargylamine, as the second amidation reagent effectively introduces an alkyne functional group into α2,3-linked sialic acid glycoproteins. In tissues, serum, and cultured cells, this allows for the detection and visualization of Nlinked glycan sialic acid isomers by imaging mass spectrometry approaches. Formalin-fixed paraffinembedded prostate cancer tissues and pancreatic cancer cell lines were used to characterize the numbers and distribution of alkyne-modified α2,3-linked sialic acid N-glycans. An azide-amine compound with a poly(ethylene glycol) linker was evaluated for use in histochemical staining. Formalin-fixed pancreatic cancer tissues were amidated with the azide amine, reacted with biotin-alkyne and copper catalyst, and sialic acid isomers detected by streptavidin-peroxidase staining. The direct chemical introduction of bioorthogonal click chemistry reagents into sialic acid-containing glycans and glycoproteins provides a new glycomic tool set to expand approaches for their detection, labeling, visualization, and enrichment.

No abstract

While mammograms are the standard tool for breast cancer screening, there remains challenges for mammography to effectively distinguish benign lesions from breast cancers, leading to many unnecessary biopsy procedures. A blood-based biomarker could provide a minimally invasive supplemental assay to increase the specificity of breast cancer screening. Serum N-glycosylation alterations have associations with many cancers and several of the clinical characteristics of breast cancer. The current study utilized a high-throughput mass spectrometry workflow to identify serum N-glycans with differences in intensities between patients that had a benign lesion from patients with breast cancer. The overall N-glycan profiles of the two patient groups had no differences, but there were several individual N-glycans with significant differences in intensities between patients with benign lesions and ductal carcinoma in situ (DCIS). Many N-glycans had strong associations with age and/or body mass index, but there were several of these associations that differed between the patients with benign lesions and breast cancer. Accordingly, the samples were stratified by the patient’s age and body mass index, and N-glycans with significant differences between these subsets were identified. For women aged 50–74 with a body mass index of 18.5–24.9, a model including the intensities of two N-glycans, 1850.666 m/z and 2163.743 m/z, age, and BMI were able to clearly distinguish the breast cancer patients from the patients with benign lesions with an AUROC of 0.899 and an optimal cutoff with 82% sensitivity and 84% specificity. This study indicates that serum N-glycan profiling is a promising approach for providing clarity for breast cancer screening, especially within the subset of healthy weight women in the age group recommended for mammograms.