Laser desorption time-of-flight mass spectrometric analysis of transferrin precipitated with antiserum: a unique simple method to identify molecular weight variants.

Abstract

Serum transferrin precipitated with anti-transferrin serum was analysed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS). The transferrin-antibody complex in the immunoprecipitates was separated into transferrin and IgG in an acidic pH, which is the usual condition of loading on MALDI-TOFMS. Ions of IgG and other minor components were not superimposed on the transferrin ions. Transferrin isoforms with different carbohydrate contents could be identified by this simple method easier than by affinity chromatography requiring the time-consuming preparation of an insolubilized specific antibody. The transferrin isoform with a molecular weight of approximately 2.2 kDa smaller than normal transferrin, which is contained in the serum from patients with carbohydrate-deficient glycoprotein (CDG) syndrome, was identified by this method. In addition to the M(1+) ion detected using sinapinic acid as a matrix, the M(2+) and M(3+) ions of transferrin were clearly detected using alpha-cyano-4-hydroxycinnamic acid as matrix and the molecular weight heterogeneity was identified more clearly in multivalent ions than that in the M(1+) ion. The MALDI-TOF analysis of immunoprecipitates may serve as a simple and sensitive method to identify the molecular weight heterogeneity of various biological materials.