ac203336u_si_001.pdf (752.66 kB)
Examination of Glycan Profiles from IgG-Depleted Human Immunoglobulins Facilitated by Microscale Affinity Chromatography
journal contribution
posted on 2012-04-03, 00:00 authored by Martin Svoboda, Benjamin F. Mann, John A. Goetz, Milos V. NovotnyAmong 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.