Stability evaluation of immobilized peptides towards proteases by mass spectrometry

SILVANA L. GIUDICESSI; MARÍA L. SALUM; MARÍA C. MARTÍNEZ CERON; OSVALDO CASCONE; ERRA BALSELLS ROSA; SILVIA A. CAMPERI

Orlando

Simposio; 24th Internattional America Peptide Symposium; 2015

American Peptide Society

Short peptides are widely used as ligands in affinity chromatography purification of proteins. However, proteases present in the crude sample may degrade immobilized peptides, shortening the chromatographic matrix useful life. Then, peptide ligand stability must be evaluated before its use in a purification process. Commonly, verification of enzymatic stability is evaluated with the soluble peptides, which differs from the immobilized peptide behavior. Further, as the peptides to be evaluated are in solution in the reaction mixture, the study of the peptide degradation products requires purification steps before analysis. In this work we developed a method to evaluate immobilized peptide stability using MALDI-MS or ESI-MS. ChemMatrix was used as the immobilized support due to its chemical stability. This PEG-based matrix allowed peptide synthesis in organic solvents and stability peptide evaluation in aqueous solvents. 4-hydroxymethylbenzoic acid was used as the linker in order to introduce a cleavage site to release the peptides from matrix before MS analysis. The model peptide FKFRYTAHGGAGG was synthesized using the Fmoc strategy. After peptide elongation, protecting groups were removed with TFA cocktail. Peptide-beads were then incubated with solution containing trypsin or chymotrypsin. After washing, the beads peptides were detached with ammonium vapor and analyzed by MS and MS/MS. Both ESI and MALD MS allowed the detection of the hall peptides as well as their C-terminal enzymatic degradation products. Although, alpha-cyano-4-hydroxycinnamic acid matrix clusters interfered in MALDI-MS the analysis of low molecular weight products, the cis-sinapinic acid could be used instead and allowed their analysis.