Glycosylated proteins play important roles in a broad spectrum of biochemical and biological processes, and prior reports have suggested that changes in protein glycosylation occur during cancer initiation and progression. Ovarian cancer (OC) is a fatal malignancy, most commonly diagnosed after the development of metastases. Therefore, early detection of OC is key to improving survival. To this end, specific changes of the serum glycome have been proposed as possible biomarkers for different types of cancers. In this study, we extend this concept to OC. To characterize differences in total N-glycan levels, serum samples provided by 20 healthy control women were compared to those acquired from patients diagnosed with late-stage recurrent OC who were enrolled in an experimental treatment trial prior to receiving therapy (N = 19) and one month later, prior to the second treatment cycle (N = 11). Additionally, analyses of the N-glycans associated with IgG and characterization of the relative abundance levels of core vs. outer-arm fucosylation were also performed. The N-linked glycomic profiles revealed increased abundances of tri- and tetra-branched structures with varying degrees of sialylation and fucosylation and an apparent decrease in the levels of “bisecting” glycans in OC samples compared to controls. Increased levels of a-galactosylated structures were observed on N-linked glycans derived from IgG, which were independent of the presence of fucose residues. Elevated levels of outer-arm fucosylation were also identified in the OC samples. These results allowed the control samples to be distinguished from the baseline ovarian cancer patients prior to receiving the experimental treatment. In some cases, the pre-treatment samples could be distinguished from the post-experimental treatment samples, as many of those patients showed a further progression of the disease.
Colorectal cancer is the fourth most prevalent cancer in the United States, yet there are no reliable non-invasive early screening methods available. Serum-based glycomic profiling has the necessary sensitivity and specificity to distinguish disease states and provide diagnostic potential for this deadly form of cancer. We applied microchip electrophoresis and MALDI-TOF-MS-based glycomic procedures to 20 control serum samples and 42 samples provided by patients diagnosed with colorectal cancer. Within the identified glycans, the position of fucose units was located to quantitate possible changes of fucosyl isomeric species associated with the pathological condition. MALDI-MS data revealed several fucosylated tri- and tetra-antennary glycans which were significantly elevated in their abundance levels in the cancer samples and distinguished the control samples from the colorectal cancer cohort in the comprehensive profiles. When compared to other cancers studied previously, some unique changes appear to be associated with colorectal cancer, being primarily associated with fucosyl isomers. Through MS and microchip electrophoresis-based glycomic methods, several potential biomarkers were identified to aid in the diagnosis and differentiation of colorectal cancer. With its unique capability to resolve isomers, microchip electrophoresis can yield complementary analytical information to MS-based profiling.
Among the most important proteins involved in the disease and healing processes are the immunoglobulins (Igs). Although many of the Igs have been studied through proteomics, aside from IgG, immunoglobulin carbohydrates have not been extensively characterized in different states of health. It seems valuable to develop techniques that permit us to understand changes in the structures and abundances of Ig glycans in the context of disease onset and progression. We have devised a strategy for characterization of the glycans for the Ig classes other than IgG (i.e. A, D, E, and M) that contain kappa light chains, while using only a few microliters of biological material. First, we designed a microcolumn containing the recombinant Protein L that was immobilized on macroporous silica particles. A similarly designed Protein G microcolumn was utilized to first perform an on-line depletion of the IgG from the sample, human blood serum, and thereby facilitate enrichment of the other Igs. While only 3 μL of serum were used in these analyses, we were able to recover a significantly-enriched fraction of non-IgG immunoglobulins. The enrichment properties of the Protein L column were characterized using a highly sensitive label-free quantitative proteomics LC-MS/MS approach, and the glycomic profiles of enriched immunoglobulins were measured by MALDI-TOF-MS. As a proof-of-principle, a comparative study was conducted using blood serum from a small group of lung cancer patients and a group of age-matched cancer-free individuals to demonstrate that the method is suitable for investigation of glycosylation changes in disease. The results were in agreement with a glycomic investigation of whole blood serum from a much larger lung cancer cohort.
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