Use of fullerene-, octadecyl-, and triaconthyl silica for solid phase extraction of tryptic peptides obtained from unmodified and in vitro glycated human serum albumin and fibrinogen.

SPE plays a crucial role in bioanalytical research. In the present work a novel fullerene(C60)-derivatised silica material is compared with octadecyl(C18) - and triaconthyl(C30)-silicas regarding recoveries of peptides and sequence coverage of HSA and fibrinogen digests. C30- and C60(30 nm)-SPE materials were found to be the two most prominent SPE materials. At low peptide concentrations C60-material prepared from a silica gel with a pore size of 30 nm has proven to be the best material with regards to recoveries. By increasing the amount of loaded peptides recoveries decrease due to its relative low binding capacity in contrast to C30-silica particles, showing no changes. The best sequence coverages of Aalpha- and Bbeta-chains of 20 pmol fibrinogen digest can also be achieved using these two SPE materials, C60 (30 nm) demonstrates an outstanding value of sequence coverage (62.15%) achieved for the gamma-chain. After nonenzymatic glycation the digests of fibrinogen and HSA were also separated. This makes the detection of a considerably higher number of glycated peptides possible compared to the unfractionated digests and the use of boronate affinity chromatography in the case of fibrinogen. For HSA, ten new sites of glycation at lysine and arginine residues have been explored. Using the detailed SPE/off-line MALDI method the glycation sites on fibrinogen are first described in this paper.