Mass spectrometric analysis of affinity-captured proteins on a dendrimer-based immunosensing surface: investigation of on-chip proteolytic digestion

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The monolayer of fourth-generation poly(amidoamine) dendrimers was adopted to construct the immunoaffinity 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 affinity-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-flight 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 buffer, and the amount of affinity-captured protein on a surface. Addition of a water-miscible organic solvent to a reaction buffer had no significant effect on the digestion efficiency under the optimized digestion conditions. The on-chip digestion method identified the affinity-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 ranging from low- to high-femtomole levels, whereas the efficiency of in-solution digestion decreased significantly as the amount of protein decreased to low-femtomole levels. The structural alignment of the peptide fragments from on-chip-digested BSA revealed that the limited exterior of the captured protein is subjected to attack by trypsin. The established detection procedures enabled the identification of BSA in the biological mixtures at the level of 0.1 ng/mL. The use of antibodies against the proteins involved in the metabolic pathway Of L-threonine in Escherichia coli also led to discrimination of the respective target proteins from cell lysates. (C) 2004 Elsevier Inc. All rights reserved.
Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
Issue Date
2005-02
Language
English
Article Type
Article
Keywords

BIOMOLECULAR INTERACTION ANALYSIS; ANTIBODY IMMOBILIZATION; DNA MICROARRAYS; MONOLAYERS; BIOSENSOR; SERUM; GOLD; IDENTIFICATION; INTEGRATION; MIXTURES

Citation

ANALYTICAL BIOCHEMISTRY, v.337, no.2, pp.294 - 307

ISSN
0003-2697
DOI
10.1016/j.ab.2004.10.042
URI
http://hdl.handle.net/10203/14289
Appears in Collection
BS-Journal Papers(저널논문)
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