Examination of Glycan Profiles from IgG-Depleted Human Immunoglobulins Facilitated by Microscale Affinity Chromatography

Abstract

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.

Complementary Glycomic Analyses of Sera Derived from Colorectal Cancer Patients by MALDI-TOF-MS and Microchip Electrophoresis

“…Based on our previous studies, 10, 49 three remaining glycans have all been tentatively associated with the various immunoglobulins. In our previous cancer glycomic studies, we, along with others, have observed a general trend of decreased abundances of galactosylated glycans and elevated levels of those carbohydrates lacking this particular monosaccharide.…”

Recent Advances in the Analysis of Complex Glycoproteins

“…23 In a similar fashion, immunoglobulins and their associated glycans can be probed in different states of health after their sequential purification through immobilized Protein G and Protein L microscale affinity columns. 36 …”

New Trends in the Study of Protein Glycosylation in Oncological Diseases

“…Třetí přístup vhodný pro účely glykoproteomiky je použití komerčně vyráběných mikrokolon Nukleosil 4000-7 (Macherey-Nagel GmbH & Co., Germany), které fungují na principu aniontového výmě-níku pro separaci proteinů a peptidů. Vhodnost mikrokolon je dána přede-vším chemickou inertností vůči zkoumanému vzorku (nedochází k nespecifi cké adsorpci jiných proteinů), kompaktností při užití HPLC (high performance liquid chromatography) a v neposlední řadě i velkým poměrem povrchu k objemu i pevností SiO 2 [49].…”