Abstract
Our group has recently developed the GlycoTyper assay which is a streamlined antibody capture slide array approach to directly profile N-glycans of captured serum glycoproteins including immunoglobulin G (IgG). This method needs only a few microliters of serum and utilizes a simplified processing protocol that requires no purification or sugar modifications prior to analysis. In this method, antibody captured glycoproteins are treated with peptide N-glycosidase F (PNGase F) to release N-glycans for detection by MALDI imaging mass spectrometry (IMS). As alterations in N-linked glycans have been reported for IgG from large patient cohorts with fibrosis and cirrhosis, we utilized this novel method to examine the glycosylation of total IgG, as well as IgG1, IgG2, IgG3 and IgG4, which have never been examined before, in a cohort of 106 patients with biopsy confirmed liver fibrosis. Patients were classified as either having no evidence of fibrosis (41 patients with no liver disease or stage 0 fibrosis), early stage fibrosis (10 METAVIR stage 1 and 18 METAVIR stage 2) or late stage fibrosis (6 patients with METAVIR stage 3 fibrosis and 37 patients with METAVIR stage 4 fibrosis (cirrhosis)). Several major alterations in glycosylation were observed that classify patients as having no fibrosis (sensitivity of 92% and a specificity of 90%), early fibrosis (sensitivity of 84% with 90% specificity) or significant fibrosis (sensitivity of 94% with 90% specificity).
Highlights
Cirrhosis is the result of chronic liver injury and leads to replacement of the normal liver architecture by fibrotic scar tissue, and is associated with a concomitant decline of liver function and devastating clinical complications [1]
Slides are washed after protein capture using a mass spectrometry compatible detergent before glycans are released by application of a thin coating of recombinant PNGase F PRIMETM to the slide (Figure 1C)
The spatial localization provided by the imaging mass spectrometry (IMS) data link the detected glycans to their corresponding captured glycoproteins
Summary
Cirrhosis is the result of chronic liver injury and leads to replacement of the normal liver architecture by fibrotic scar tissue, and is associated with a concomitant decline of liver function and devastating clinical complications [1]. In the case of liver fibrosis, two analytical methods have identified alterations in N-linked glycosylation on both total IgG populations and on specific IgG molecules Both methods can detect cirrhosis with a high degree of accuracy and are able to detect intermediate levels of fibrosis [11, 16]. One approach utilizes a capillary electrophoresis-based analysis of N-linked glycans on total serum following release of glycans using PNGase F, labeling of the released glycans using a fluorescent dye, and electrophoresis of the released glycans for peak identification [11] While this method works well, it is both time and labor intensive. We have used the GlycoTyper to demonstrate its ability to measure changes in the N-linked glycans on IgG subtypes and serve as a surrogate marker of liver disease
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