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Development and characterization of cIEF‐MALDI‐TOF MS for protein analysis
Author(s) -
Silvertand Linda H. H.,
Toraño Javier Sastre,
de Jong Gerhardus J.,
van Bennekom Wouter P.
Publication year - 2009
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200800740
Subject(s) - electropherogram , chemistry , chromatography , spotting , isoelectric focusing , fractionation , sample preparation , analytical chemistry (journal) , resolution (logic) , electrophoresis , artificial intelligence , computer science , enzyme , biochemistry
This paper describes the hyphenation of cIEF and MALDI‐TOF MS via a fractionation or spotting device. After focusing in cIEF the compounds are hydrodynamically mobilized and deposited on a MALDI target plate using a sheath liquid interface, which provides the catholyte solution and the electrical ground. From previous experiments, sample conditions that resulted in a high resolution in cIEF and acceptable protein signal intensity in MS were selected [Silvertand et al ., Electrophoresis , 2008, 29 , 1985–1996]. Besides the mixture of test proteins, the sample solution contains 1% Pharmalyte, 0.3% hydroxyethyl cellulose and 0.1% Tween 20 and is used for both optimization as well as characterization of the cIEF‐MALDI‐TOF MS system. Hyphenation problems encountered are mainly due to transfer of the liquid from the needle to the MALDI target plate and are solved by choosing the proper sheath catholyte (200 mM NH 4 OH in 50% methanol with 0.1% Tween20). MS electropherograms were reconstructed by plotting the intensities of the m/z values corresponding to the proteins versus migration time (related to spot number). Reproducibility, peak width and signal intensity for different focusing and spotting (fractionation) times were calculated using these reconstructed MS electropherograms as well as the UV electropherograms. The best results were obtained with focusing time of 75 min (no under‐ or overfocusing) and a spotting time of 5 s (highest protein signal intensity in MS). The applicability of the system is demonstrated by the analysis of a biopharmaceutical (glucagon) and its deamidation product.