Stability Evaluation of Immobilized Peptides towards Proteases by Mass Spectrometry

S.L. GIUDICESSI, ; M.L. SALUM, ; M.C. MARTÍNEZ-CERON ; O. CASCONE, ; S.A. CAMPERI,; ERRA-BALSELLS, R.; R, ERRA BASELLS,

Orlando

Simposio; American peptide symposium 2015; 2015

American Ppetide Society

Short peptides are widely used as ligands inaffinity chromatography purification of proteins. However, proteases present inthe crude sample may degrade immobilized peptides, shortening thechromatographic matrix useful life. Then, peptide ligand stability must beevaluated before its use in a purification process. Commonly, enzymaticstability is evaluated with soluble peptides, which differs from theimmobilized peptide behavior. Further, the study of the peptide degradationproducts requires purification steps before analysis. In this work we developeda method to evaluate immobilized peptide stability using MALDI-MS or ESI-MS.ChemMatrix was used as immobilized support due to its chemical stability. ThisPEG-based matrix allowed peptide synthesis in organic solvents and stabilitypeptide evaluation in aqueous solvents. 4-hydroxymethylbenzoic acid was used aslinker. The model peptide FKFRYTAHSGASG was synthesized using the Fmocstrategy. Peptide-beads were then incubated with solution containing proteases.After washing, the beads peptides were detached with ammonia vapor and analyzedby MS and MS/MS. Both ESI-MS and MALDI-MS allowed the detection of the hallpeptides as well as their C-terminal enzymatic degradation products. Althoughalpha-cyano-4-hydroxycinnamic acid matrix clusters interfered in MALDI-MSanalysis of low molecular weight products, cis-sinapinic acid matrix allowed theiranalysis.