One-bead, one-compound peptide library sequencing via high-pressure ammonia cleavage coupled to nanomanipulation/nanoelectrospray ionization mass spectrometry.
Biological screening of one-bead, one-compound (OBOC) combinatorial peptide libraries is routinely carried out with the peptide remaining bound to the resin bead during screening. After a hit is identified, the bead is isolated, the peptide is cleaved from the bead, and its sequence is determined. We have developed a new technique for cleavage of peptides from resin beads whereby exposure of a 4-hydroxymethyl benzoic acid (HMBA)-linked peptide to high-pressure ammonia gas led to efficient cleavage in as little as 5min. Here we also report a new method of extracting peptide from individual library beads for its introduction into a mass spectrometer that uses nanomanipulation combined with nanoelectrospray ionization mass spectrometry (NSI MS). Single beads analyzed by nanomanipulation/NSI MS were found to give identical MS results to those of bulk samples. Detection of 18 unique cleaved peptides 1 to 8 amino acids in length, and sequencing of 14 different peptide sequences 4 to 8 amino acids in length, was demonstrated on a combination of bulk samples and ones from individual beads of an OBOC library. The method was highly reproducible, with 100% of attempts to extract peptide resulting in high-quality MS data. This new collection of techniques allows rapid, reliable, environmentally responsible sequencing of hit beads from combinatorial peptide libraries.
Brown
Jennifer M
JM
Department of Chemistry, University of North Texas, Denton, TX 76203, USA.
Hoffmann
William D
WD
Alvey
Cory M
CM
Wood
Addison R
AR
Verbeck
Guido F
GF
Petros
Robby A
RA
D000595:Amino Acid Sequence; D000641:Ammonia / Q000737:chemistry*; D020650:Combinatorial Chemistry Techniques; D036103:Nanotechnology / Q000379:methods*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry*; D011312:Pressure; D012117:Resins, Synthetic / Q000737:chemistry; D020539:Sequence Analysis, Protein / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D019151:Peptide Library; D010455:Peptides; D012117:Resins, Synthetic; D000641:Ammonia