One-bead, one-compound peptide library sequencing via high-pressure ammonia cleavage coupled to nanomanipulation/nanoelectrospray ionization mass spectrometry

• Published in: Analytical biochemistry Vol. 398; no. 1; pp. 7 - 14
• Main Authors: Brown, Jennifer M., Hoffmann, William D., Alvey, Cory M., Wood, Addison R., Verbeck, Guido F., Petros, Robby A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-03-2010
• Subjects: Amino Acid Sequence; Ammonia - chemistry; Combinatorial Chemistry Techniques; HMBA; Mass spectrometry; Nanotechnology - methods; OBOC peptide library; Peptide cleavage; Peptide Library; Peptides - chemistry; Pressure; Resins, Synthetic - chemistry; Sequence Analysis, Protein - methods; Sequencing; Spectrometry, Mass, Electrospray Ionization - methods; Peptide cleavage; OBOC peptide library; HMBA; Sequencing; Mass spectrometry
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2009.10.044

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 5
min. 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.