Fluorescence-based peptide screening using ligand peptides directly conjugated to a thiolated glass surface.
Functional peptides from peptide libraries are frequently screened using an array format. We report here results of a feasibility study of fluorescence-based peptide screening using an array format on surface-modified glass. The surface of an amine-coated glass slide was modified to contain thiol groups by iminothiolane treatment. The epsilon-amine of the C-terminal lysine from a ligand peptide was iodinated and then spotted onto the thiolated glass surface to covalently conjugate the ligand peptide to the surface via a thioether bond. This covalent immobilization allowed the ligand peptides to withstand washing steps by tightly adhering to the glass surface and confining their subsequent binding reactions within a spotted area. Two representative peptides were used as the ligand peptides; a 'target' (positive) heptapeptide that could specifically bind to trypsin, and a 'control' (negative) hexapeptide that had no binding affinity with trypsin. When fluorescein isothiocyanate-labeled trypsin was reacted with the ligand peptides, the target peptide demonstrated distinctively higher (ca. 8.7-fold) fluorescence intensity that was easily differentiated from the control peptide by a fluorescence scanner. A separate experiment using a quartz crystal microbalance confirmed that the difference in binding mass (ca. 9.1-fold) was very close to that seen in fluorescence intensity. These results suggested a quantitative, 1:1 correlation between mass and fluorescence signals. Furthermore, a smaller spot volume and a higher ligand peptide concentration resulted in higher fluorescence signal intensity. This study provides information on the potential for using fluorescence-based screening of functional peptides on a glass array format.
Lim
Chang Hwan
CH
Bioprocessing Research Laboratory, Department of Bionanotechnology, Hanyang University, Ansan, Korea.
Cho
Hyung Min
HM
Choo
Jaebum
J
Neff
Silke
S
Jungbauer
Alois
A
Kumada
Yoichi
Y
Katoh
Shigeo
S
Lee
Eun Kyu
EK
D015374:Biosensing Techniques; D005240:Feasibility Studies; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005898:Glass / Q000737:chemistry; D006046:Gold / Q000737:chemistry; D054879:Halogenation; D008024:Ligands; D008956:Models, Chemical; D015394:Molecular Structure; D008970:Molecular Weight; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D011791:Quartz / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D005456:Fluorescent Dyes; D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D013438:Sulfhydryl Compounds; D011791:Quartz; D014443:Tyrosine; D006046:Gold; D016650:Fluorescein-5-isothiocyanate