Identification of protein-binding peptides by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of peptide beads selected from the screening of one bead-one peptide combinatorial libraries.
A fast and inexpensive strategy for the identification of peptide ligands by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of peptide beads screened from one bead-one peptide combinatorial libraries is herein described. Streptavidin was used as the model protein. A combinatorial library of 6561 peptides was synthesized on ChemMatrix resin by the divide-couple-recombine method. 4-Hydroxymethylbenzoic acid was used as the linker and five residues of Gly were incorporated at the C termini to increase the final peptide molecular weight. Positive control peptides with the HPQ motif and negative control peptides without the HPQ motif evidenced that the linker and the five residues of Gly have neither impaired the specific binding nor facilitated unspecific binding. After screening the library, positive beads were isolated and washed with 8M guanidine hydrochloride. The beads were sliced into two or four pieces, deposited onto the stainless steel MALDI sample plate, and treated with ammonia vapor to release the peptides. In addition, 26 beads picked at random from the library were subjected to the same treatment. All samples were analyzed by MALDI-TOF-MS and the peptides were unambiguously identified with very good reproducibility between the bead pieces, thus evidencing the good homogeneity of the bead. All sequences obtained from the screening contained HPQ.
Marani
Mariela M
MM
Institute for Research in Biomedicine, Barcelona Science Park, Josep Samitier 1, 08028 Barcelona, Spain.
Oliveira
Eliandre
E
Côte
Simón
S
Camperi
Silvia A
SA
Albericio
Fernando
F
Cascone
Osvaldo
O
D001665:Binding Sites; D013058:Mass Spectrometry; D009842:Oligopeptides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D009842:Oligopeptides; D010455:Peptides; D011506:Proteins