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Mass Spectrometric Analysis of Affinity-captured Proteins on a Dendrimer-based Immunosensing Surface: Investigation of On-chip Proteolytic Digestion by Hak-Joon Seok; Mi-Young Hong; Young-Ja Kim; Min-Kyu Han; Dohoon Lee; Jung-Hwa Lee; Jong-Shin Yoo; Hak-Sung Kim is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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The monolayer of fourth-generation poly(amidoamine) dendrimers was adopted to construct the immunoaYnity surface of an antibody layer. The antibody layer as a bait on the dendrimer monolayer was found to result in high binding capacity of antigenic proteins and a reliable detection. The aYnity-captured protein at the immunosensing surface was subjected to direct on-chip tryptic digestion, and the resulting proteolytic peptides were analyzed by using matrix-assisted laser desorption/ionization time-of-Xight mass spectrometry. The performance of the on-chip digestion procedure was investigated with respect to the ratio of trypsin to protein, digestion time, composition of a reaction buVer, and the amount of aYnity-captured protein on a surface. Addition of a water-miscible organic solvent to a reaction buVer had no signiWcant eVect on the digestion eYciency under the optimized digestion conditions. The on-chip digestion method identiWed the aYnity-captured bovine serum albumin (BSA), lysozyme, and ferritin at the level of around 100 fmol. Interestingly, the detected number of peptide hits through the on-chip digestion was almost similar regardless of the amount of captured protein ra

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Author

Hak-Joon Seok; Mi-Young Hong; Young-Ja Kim; Min-Kyu Han; Dohoon Lee; Jung-Hwa Lee; Jong-Shin Yoo; Hak-Sung Kim

Publisher

Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)

Published

2005

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)