The work described here examines displacement phenomena that play a role in
lectin affinity chromatography and their potential to impact reproducibility. This was achieved using
Lycopersicon esculentum lectin (LEL), a
lectin widely used in monitoring
cancer. Four small identical LEL columns were coupled in series to form a single affinity chromatography system with the last in the series connected to an absorbance detector. The serial affinity column set (SACS) was then loaded with human
plasma proteins. At the completion of loading, the column set was disassembled, the four columns were eluted individually, the captured
proteins were
trypsin digested, the
peptides were deglycosylated with
PNGase F, and the parent
proteins were identified through mass spectral analyses. Significantly different sets of
glycoproteins were selected by each column, some
proteins appearing to be exclusively bound to the first column while others were bound further along in the series. Clearly, sample displacement chromatography (SDC) occurs.
Glycoproteins were bound at different places in the column train, identifying the presence of glycoforms with different affinity on a single
glycoprotein. It is not possible to see these phenomena in the single column mode of chromatography. Moreover, low abundance
proteins were enriched, which facilitates detection. The great advantage of this method is that it differentiates between
glycoproteins on the basis of their binding affinity. Displacement phenomena are concluded to be a significant component of the separation mechanism in heavily loaded
lectin affinity chromatography columns. This further suggests that care must be exercised in sample loading of
lectin columns to prevent analyte displacement with nonretained
proteins.