Protein prenylation is a post-translational modification where a 15 carbon farnesyl or 20 carbon geranylgeranyl isoprenoid is appended to the C terminal end of a protein by either farnesyltransferase (FTase) or geranylgeranyl transferase type 1, respectively. In the canonical understanding of Farnesyltransferase, the isoprenoids are attached to the Cysteine residue of a four amino acid CaaX box sequence. It was widely believed that the Farnesyltransferase could only recognize this four amino acid sequence. However, recent work has shown that five amino acid sequences can also be recognized, such as the pentapeptide CMIIM. This new discovery greatly increases the number of potential FTase substrates, as the FTase is already known to tolerate a wide variety of amino acids in the canonical CaaX box. However, traditional methods to evaluate a wide variety of potential substrates are not without flaws, with many relying on randomization of the amino acids in the CaaX box, or a series of reactions with a bioorthogonal probe to allow for fluorophore conjugation. With the goal of developing a more rapid and methodical method to evaluate potential substrates, we envisioned using MALDI-MS to quickly and easily assay complex peptide libraries with the goal of varying one amino acid in the CaaX box to all 20 canonical amino acids. We used this strategy to examine the previously discovered CMIIM peptide, modifying each amino acid position one at a time to all 20 amino acids to probe substrate space for similar peptides. Through this method we observed 30 hits in the mass spectrum, following up on eight of them with HPLC experiments. We determined that seven of our peptides were substrates for FTase with a similar efficiency to the parent CMIIM. Studies that probe the prenylation of these sequences in living cells are in progress.

MALDI-MS as a tool to rapidly screen peptide libraries for novel substrates of farnesyltransferase

ACS Spring Meeting 2021

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