Mass spectrometric analysis of affinity-captured proteins on a dendrimer-based immunosensing surface: investigation of on-chip proteolytic digestion.
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.
Seok
Hak-Joon
HJ
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Kusung-dong, Yusung-gu, Daejon 305-701, Republic of Korea.
Hong
Mi-Young
MY
Kim
Young-Ja
YJ
Han
Min-Kyu
MK
Lee
Dohoon
D
Lee
Jung-Hwa
JH
Yoo
Jong-Shin
JS
Kim
Hak-Sung
HS
D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods*; D002417:Cattle; D004926:Escherichia coli; D005293:Ferritins / Q000032:analysis / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000032:analysis / Q000378:metabolism; D011108:Polymers / Q000737:chemistry*; D040081:Protein Array Analysis / Q000295:instrumentation*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism*
D011108:Polymers; D011506:Proteins; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine; D005293:Ferritins; D009113:Muramidase; D014357:Trypsin