Stability evaluation of immobilized peptides towards proteases by mass spectrometry

SILVANA L. GIUDICESSI; MARÍA L. SALUM; CAMILA MARTINEZ CERON; O CASCONE; ROSA ERRA-BALSELLS; SILVIA A. CAMPERI

Proceedings of the 24th American Peptide Symposium

Prompt Scientific Publishing

Lugar: San Diego; Año: 2015; p. 131 - 133

Short peptides are widely used as ligands in affinity chromatography purification of proteins. However, peptidases and proteases present in the crude sample may degrade immobilized peptides, shortening the affinity support useful life. Commonly, enzymatic stability is evaluated with the peptide in solution, which may differ from the resin-bound peptide behavior. In this work we developed a strategy to evaluate immobilized peptide stability using electrospray ionization (ESI) or matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS). We used two matrices for MS analyses: a) alfa-cyano-4-hydroxycinnamic acid (CHCA) and b) Z-sinapinic acid (SA). Mass spectra were acquired in the MS reflector positive ion mode. When analyzing the peptide and their degradation products with MALDI-MS, CHCA clusters interfered in MALDI-MS analysis of low molecular weight products. On the other hand, SA matrix allowed their analysis. The method here developed allowed a fast evaluation of peptide ligands stability in solid phase towards the proteases that may be present in the crude sample before their use in affinity chromatography. Due to the high sensitive of mass spectrometry, only a small sample of peptidyl-resin is required to evaluate its stability. As the enzymatic degradation is performed in solid-phase, none hard purification protocols are needed before analysis.