Development and characterization of cIEF‐MALDI‐TOF MS for protein analysis

Abstract

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 NH4OH 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.

Capillary isoelectric focusing coupled offline to matrix assisted laser desorption/ionization mass spectrometry

“…Baseline resolution between β-lactoglobulin A (pI 5.0; MW 18,360 Da) and β-lactoglobulin B (pI 5.3; MW 18,270 Da) was only observed when using the shallower pH gradient 5-8. The MS signals likely are subject to ampholyte suppression although the extent to which was not examined [6, 13]. Overall, MALDI-MS protein detection extends the applicability of cIEF by improving detection limits relative to absorbance measurement.…”

“…A key candidate that can contribute to the analysis of protein variance is capillary isoelectric focusing (cIEF) coupled offline to matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The coupling of cIEF with MALDI-MS has been only demonstrated a few times [1-6] and is in need of further development in order to reach its full potential. The technical details presented here provide a basis for expanding the use of the technique to solve greater proteomic/analytical problems.…”

Application of Micro-Fluidic Devices for Biomarker Analysis in Human Biological Fluids

“…proteins and peptides. (Silvertand et al, 2009) Micellar microemulsion electrokinetic chromatography (MEEKC) is a mode of CE, which utilizes microemulsions as separation media and allows for separation of neutral as well as charged analytes. (Ryan et al, 2010) Capillary electrochromatography is a hybrid of CE and high performance liquid chromatography, which provides both selectivity and efficiency.…”

Sequential injection setup for capillary isoelectric focusing combined with MS detection

“…The first coupling of MS and CIEF [18] showed the advantage of using IEF-MS over common CZE-MS, since the limit of detection was lower in the former case. Several publications showed the usefulness of this coupling for the analysis of proteins from different physiological fluids or tissues [19][20][21][22][23][24], as well as the application of free-flow electrophoresis approach [16,25], and also the two-dimensional offline separation setup of CIEF and MALDI-TOF-MS analyses [26][27][28][29][30][31]. Despite the promising results, the classical problems, i.e.…”