Metabolic Markers and Association of Biological Sex in Lupus Nephritis.

Lupus nephritis (LN) is a serious complication for many patients who develop systemic lupus erythematosus, which primarily afflicts women. Our studies to identify biomarkers and the pathogenic mechanisms underlying LN will provide a better understanding of disease progression and sex bias, and lead to identification of additional potential therapeutic targets. The glycosphingolipid lactosylceramide (LacCer) and N-linked glycosylated proteins (N-glycans) were measured in urine and serum collected from LN and healthy control (HC) subjects (10 females and 10 males in each group). The sera from the LN and HC subjects were used to stimulate cytokine secretion and intracellular Ca2+ flux in female- and male-derived primary human renal mesangial cells (hRMCs). Significant differences were observed in the urine of LN patients compared to HCs. All major LacCers species were significantly elevated and differences between LN and HC were more pronounced in males. 72 individual N-glycans were altered in LN compared to HC and three N-glycans were significantly different between the sexes. In hRMCs, Ca2+ flux, but not cytokine secretion, was higher in response to LN sera compared to HC sera. Ca2+ flux, cytokine secretion, and glycosphingolipid levels were significantly higher in female-derived compared to male-derived hRMCs. Relative abundance of some LacCers and hexosylceramides were higher in female-derived compared to male-derived hRMCs. Urine LacCers and N-glycome could serve as definitive LN biomarkers and likely reflect renal disease activity. Despite higher sensitivity of female hRMCs, males may experience greater increases in LacCers, which may underscore worse disease in males. Elevated glycosphingolipid metabolism may poise renal cells to be more sensitive to external stimuli.

Bioorthogonal Chemical Labeling Probes Targeting Sialic Acid Isomers for N-Glycan MALDI Imaging Mass Spectrometry of Tissues, Cells, and 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 α2,3- and 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 N-linked glycan sialic acid isomers by imaging mass spectrometry approaches. Formalin-fixed paraffin-embedded 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.

Analysis of N-linked Glycan Alterations in Tissue and Serum Reveals Promising Biomarkers for Intrahepatic Cholangiocarcinoma.

There is an urgent need for the identification of reliable prognostic biomarkers for patients with intrahepatic cholangiocarcinoma (iCCA) and alterations in N-glycosylation have demonstrated an immense potential to be used as diagnostic strategies for many cancers, including hepatocellular carcinoma (HCC). N-glycosylation is one of the most common post-translational modifications known to be altered based on the status of the cell. N-glycan structures on glycoproteins can be modified based on the addition or removal of specific N-glycan residues, some of which have been linked to liver diseases. However, little is known concerning the N-glycan alterations that are associated with iCCA. We characterized the N-glycan modifications quantitatively and qualitatively in three cohorts, consisting of two tissue cohorts: a discovery cohort (n = 104 cases) and a validation cohort (n = 75), and one independent serum cohort consisting of patients with iCCA, HCC, or benign chronic liver disease (n = 67). N-glycan analysis in situ was correlated to tumor regions annotated on histopathology and revealed that bisected fucosylated N-glycan structures were specific to iCCA tumor regions. These same N-glycan modifications were significantly upregulated in iCCA tissue and serum relative to HCC and bile duct disease, including primary sclerosing cholangitis (PSC) (P < 0.0001). N-glycan modifications identified in iCCA tissue and serum were used to generate an algorithm that could be used as a biomarker of iCCA. We demonstrate that this biomarker algorithm quadrupled the sensitivity (at 90% specificity) of iCCA detection as compared with carbohydrate antigen 19-9, the current "gold standard" biomarker of CCA. Significance: This work elucidates the N-glycan alterations that occur directly in iCCA tissue and utilizes this information to discover serum biomarkers that can be used for the noninvasive detection of iCCA.

Integrating age, BMI, and serum N-glycans detected by MALDI mass spectrometry to classify suspicious mammogram findings as benign lesions or breast cancer.

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.

Direct N-Glycosylation Profiling of Urine and Prostatic Fluid Glycoproteins and Extracellular Vesicles.

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.

Glycan Imaging Mass Spectrometry: Progress in Developing Clinical Diagnostic Assays for Tissues, Biofluids, and Cells.

N-glycan imaging mass spectrometry (IMS) can rapidly and reproducibly identify changes in disease-associated N-linked glycosylation that are linked with histopathology features in standard formalin-fixed paraffin-embedded tissue samples. It can detect multiple N-glycans simultaneously and has been used to identify specific N-glycans and carbohydrate structural motifs as possible cancer biomarkers. Recent advancements in instrumentation and sample preparation are also discussed. The tissue N-glycan IMS workflow has been adapted to new glass slide-based assays for effective and rapid analysis of clinical biofluids, cultured cells, and immunoarray-captured glycoproteins for detection of changes in glycosylation associated with disease.

Rapid N-Glycan Profiling of Serum and Plasma by a Novel Slide-Based Imaging Mass Spectrometry Workflow.

Changes in the levels and compositions of N-glycans 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.