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Downsizing proteolytic digestion and analysis using dispenser‐aided sample handling and nanovial matrix‐assisted laser/desorption ionization‐target arrays
Author(s) -
Ericsson David,
Ekström Simon,
Nilsson Johan,
Bergquist Jonas,
MarkoVarga György,
Laurell Thomas
Publication year - 2001
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/1615-9861(200109)1:9<1072::aid-prot1072>3.0.co;2-w
Subject(s) - chemistry , lysozyme , chromatography , matrix assisted laser desorption/ionization , substrate (aquarium) , desorption , analytical chemistry (journal) , mass spectrometry , sample preparation , matrix (chemical analysis) , volume (thermodynamics) , biochemistry , oceanography , adsorption , geology , physics , quantum mechanics
An efficient technique for enzymatic digestion of proteins in nanovial arrays and identification by peptide mass fingerprinting using matrix‐assisted laser desorption/ionization (MALDI‐MS) is presented in this work. Through dispensing of a protein solution with simultaneous evaporation the protein (substrate) is concentrated up to 300 times in‐vial. At higher substrate concentrations the catalytic turnover numbers increase according to the Michaelis‐Menten kinetics. Therefore, the dispenser‐aided nanodigestion is valuable for identification of low‐level proteins (10 n M –500 n M ) as well as for automatic high efficiency digestions performed in 0.2–10 min. As an example of low‐level protein identification, a 10 n M solution of lysozyme C was unambiguously identified after 5 min of nanodigestion. Moreover, only 30 s nanodigestion was sufficient to identify hemoglobin (10 ν M ), exemplifying the fast catalysis of the nanodigestion technique. The developed silicon flow‐through piezoelectric dispenser is adapted for low‐volume and preconcentrated samples in the nL‐νL 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 explored. Furthermore, the potential of automated protein identification using precoated proteolytic nanovial‐arrays is demonstrated.