Abstract
Alteration in glycosylation has been observed in cancer. However, monitoring glycosylation changes during breast cancer progression is difficult in humans. In this study, we used a well-characterized transplantable breast tumor mouse model, the mouse mammary tumor virus-polyoma middle T antigen, to observe early changes in glycosylation. We have previously used the said mouse model to look at O-linked glycosylation changes with breast cancer. In this glycan biomarker discovery study, we examined N-linked glycan variations during breast cancer progression of the mouse model but this time doubling the number of mice and blood draw points. N-glycans from total mouse serum glycoproteins were profiled using matrix-assisted laser desorption/ionization Fourier transform-ion cyclotron resonance mass spectrometry at the onset, progression, and removal of mammary tumors. We observed four N-linked glycans, m/z 1339.480 (Hex(3)HexNAc), 1485.530 (Hex(3)HexNAc(4)Fuc), 1809.639 (Hex(5)HexNAc(4)Fuc), and 1905.630 (Man(9)), change in intensity in the cancer group but not in the control group. In a separate study, N-glycans from total human serum glycoproteins of breast cancer patients and controls were also profiled. Analysis of human sera using an internal standard showed the alteration of the low-abundant high-mannose glycans, m/z 1419.475, 1581.528, 1743.581, 1905.634 (Man(6-9)), in breast cancer patients. A key observation was the elevation of a high-mannose type glycan containing nine mannoses, Man(9), m/z 1905.630 in both mouse and human sera in the presence of breast cancer, suggesting an incompletion of the glycosylation process that normally trims back Man(9) to produce complex and hybrid type oligosaccharides.
Highlights
Breast cancer is the leading cause of cancer death and the most frequently diagnosed cancer among women worldwide
We have looked at N-linked glycans, i.e. glycans with a common trimannosyl chitobiose core attached to asparagine (Asn) occurring in the sequon Asn-X-Ser/Thr, where X could be any amino acid except proline (Pro), as potential bi
Omarkers for prostate cancer [18]. These N-linked glycans were released with peptide N-glycosidase F (PNGase F). In this glycan biomarker exploratory study, we monitored the N-linked glycans in a bigger sample set of the PyMT mouse model using MALDI Fourier transform ion cyclotron resonance (FT-ICR) Mass spectrometry (MS), an MS technique that we have proven to be useful in the given complex biological sample [19]
Summary
Mouse Serum Samples—Sera were collected via orbital eye bleeding of female inbred FVB mice (n ϭ 8). Three mice had only five instead of six blood samples. The cancer group (n ϭ 4) consisted of mice surgically transplanted with 1 mm pieces of highly metastatic breast tumors, polyoma middle-T Met-1, at week 0. Serum samples were collected by standard venous phlebotomy, stored in standard clot tubes and frozen at Ϫ80 °C before processing. The mixture was frozen to Ϫ80 °C for 1 h and centrifuged for 20 min at 13,200 rpm. The cartridge was conditioned with nanopure water and 80% acetonitrile (ACN) in 0.05% aqueous trifluoroacetic acid (v/v). The glycan solution was loaded onto the cartridge and desalted with nanopure water (12 ml) at a flow rate of about 1 ml/min. Each fraction was collected and dried in a centrifugal evaporator and reconstituted in nanopure water prior to mass spectrometry analysis
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