Downsizing proteolytic digestion and analysis using dispenser-aided samplehandling and nanovial matrix-assisted laser/desorption ionization-target arrays

An efficient technique for enzymatic digestion of proteins in nanovial arra ys and identification by peptide mass fingerprinting using matrix-assisted laser desorption/ionization (MALDI-MS) is presented in this work. Through d ispensing of a protein solution with simultaneous evaporation the protein ( substrate) is concentrated up to 300 times in-vial. At higher substrate con centrations the catalytic turnover numbers increase according to the Michae lis-Menten kinetics. Therefore, the dispenser-aided nanodigestion is valuab le for identification of low-level proteins (10 nM-500 nm) as well as for a utomatic high efficiency digestions performed in 0.2-10 min. As an example of low-level protein identification, a 10 nm solution of lysozyme C was una mbiguously identified after 5 min of nanodigestion. Moreover, only 30 s nan odigestion was sufficient to identify hemoglobin (10 pm), exemplifying the fast catalysis of the nanodigestion technique. The developed silicon flow-t hrough piezoelectric dispenser is adapted for low-volume and preconcentrate d samples in the nL-muL range and provides fast, accurate and contact-free sample positioning into the nanovials. In this work, the properties of the nanodigestion concept regarding proteins of different characteristics are e xplored. Furthermore, the potential of automated protein identification usi ng precoated proteolytic nanovial-arrays is demonstrated.