Examination of glycan profiles from IgG-depleted human immunoglobulins facilitated by microscale affinity chromatography

Abstract

Among the most important proteins involved in 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 an understanding of 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 that requires only a few microliters of biological material. First, we designed a microcolumn containing recombinant Protein L that was immobilized on macroporous silica particles. A similarly designed Protein G microcolumn was utilized to first perform an online depletion of the IgG from the sample, human blood serum, and thereby facilitate enrichment of the other Igs. Even though only 3 μL of serum was 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.

Figure 1

Chromatograms for (triplicate) enrichment of 500 μg of IgG on a Protein L-silica microcolumn (50 × 0.5 mm). The sample was loaded in PBS and unbound protein was washed from the column at a flow rate of 20 μL/min. After 35 min, 100 mM acetic acid was applied at a flow rate of40 μL/min to elute bound IgG.

Figure 2

Relative abundance of N-glycan structures identified in the Protein L-fractions of immunoglobulins, which were separated using an initial volume of 3 μl of pooled female serum for each analysis. Abundance was evaluated using the in-house developed program PeakCalc. Six technical replicates were performed for the affinity chromatography experiments, while each technical replicate was spotted six times on the MALDI target. The mean relative intensity from the separate enrichment experiments was used to determine the average value and standard deviation for each glycan structure. Monosaccharides are depicted as follows: blue square represents N-acetylglucosamine, green circle is mannose, yellow circle is galactose, red triangle represents fucose, and pink diamond is sialic acid.

Figure 3

A comparison of the relative intensities of the glycan structures from the IgG-depleted immunoglobulins. Six samples from cancer patients were compared to six samples from control, cancer-free individuals. Abundance was evaluated using the in-house developed program PeakCalc. Six technical replicates were performed for the affinity chromatography experiments, while each technical replicate was spotted six times on the MALDI target. The mean relative intensity from the separate enrichment experiments was used to determine the average value and standard deviation for each glycan structure. Monosaccharides depicted are the same as in Fig. 2.