Accurate MALDI-TOF/TOF sequencing of one-bead-one-compound peptide libraries with application to the identification of multiligand protein affinity agents using in situ click chemistry screening.
Combinatorial one-bead-one-compound (OBOC) peptide libraries are widely used for affinity screening, and the sequencing of peptides from hit beads is a key step in the process. For rapid sequencing, CNBr cleavage of the peptides from the beads, followed by de novo sequencing by MALDI-TOF/TOF, is explored. We report on a semiautomated sequencing algorithm and validate it through comparison against Edman degradation sequencing. The initial 44% sequencing success rate of the standard de novo sequencing software was improved to nearly 100%. The sequencing algorithm incorporates existing knowledge of amino acid chemistry and a new strategy for differentiating isobaric amino acids. We tested the algorithm by using MALDI-TOF/TOF to identify a peptide biligand affinity agent against the protein bovine carbonic anhydrase II, starting from comprehensive one-bead-one-compound peptide libraries comprised of non-natural and artificial amino acid components and using the strategy of in situ click/OBOC library screening.
Lee
Su Seong
SS
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669. sslee@ibn.a-star.edu.sg
Lim
Jaehong
J
Tan
Sylvia
S
Cha
Junhoe
J
Yeo
Shi Yun
SY
Agnew
Heather D
HD
Heath
James R
JR
D000595:Amino Acid Sequence; D000818:Animals; D024402:Carbonic Anhydrase II / Q000378:metabolism; D002417:Cattle; D008024:Ligands; D019151:Peptide Library; D010455:Peptides / Q000032:analysis*; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D024402:Carbonic Anhydrase II