Recent developments in capillary isoelectric focusing

Silvertand, L.H.H.; Toraño, Javier Sastre; Bennekom, W.P. van; Jong, Gerhardus J. de

doi:10.1016/j.chroma.2008.05.057

Cited by 77 publications

(68 citation statements)

References 143 publications

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“…Although all the separation modes, including one or two multidimensional RPLC and/or CE, have been widely used for complex samples analysis [12][13][14][15][16], CIEF, which shows excellent concentration capacity, high resolution and high sensitivity, has drawn much attention especially for the separation of proteins [17][18][19][20]. In contrast, RPLC has advantages of high efficiency and high resolution, and more importantly, the mobile phase is compatible with MS, demonstrating preferable attributes for peptide separation [21][22][23].…”

Section: Tingting Wangmentioning

confidence: 99%

Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed‐phase liquid chromatography with mass spectrometry for online protein profiling

Wang

et al. 2011

Electrophoresis

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An integrated platform with the combination of protein and peptide separation was established via online protein digestion, by which proteins were first separated by CIEF, online digested by a trypsin immobilized enzyme microreactor, trapped and desalted by two parallel trap columns, separated by nanoreversed-phase and finally identified by MS. In such a platform, two hollow fiber membrane interfaces were used. One was applied to supply catholyte and electric contact, and another to supply adjustment buffer to improve the compatibility of protein separation and tryptic digestion. A poly(octadecyl acrylate-co-ethylene dimethacrylate) monolithic column served as the trap column to capture sample and to remove the ampholytes from CIEF. A hybrid silica monolith-based immobilized trypsin microreactor was used for online protein digestion. To evaluate the performance of such a platform, a 4-protein mixture with a loading amount of only 0.29 μg, was analyzed, and sequence coverages for BSA, myoglobin, β-lactoglobulin and ribonuclease A were 8, 26, 10 and 54%, respectively. Furthermore, such an integrated platform was successfully applied for the analysis of proteins extracted from Escherichia coli, and 101 proteins were positively identified. We anticipate that the integrated platform developed herein will provide a promising tool for low-abundance protein identification with the combination of top-down and bottom-up approaches.

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Section: Tingting Wangmentioning

confidence: 99%

Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed‐phase liquid chromatography with mass spectrometry for online protein profiling

Wang

et al. 2011

Electrophoresis

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“…Although hundreds of papers have appeared, it is clear that this versatile technique for analysing amphoteric compounds has not reached that high utilisation like other capillary electrophoretic methods. Several reviews have been published on the use of this high-performance separation method [9][10][11][12].…”

Section: Introductionmentioning

confidence: 99%

Sequential injection setup for capillary isoelectric focusing combined with MS detection

Páger¹,

Dörnyei²,

Kilár³

2011

Electrophoresis

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Capillary isoelectric focusing in the presence of electroosmosis with sequential injection of carrier ampholytes and sample was found to be suitable for MS detection. The separate injection of the sample and the ampholytes provides good condition to suppress and overcome the undesirable effect of the presence of ampholytes in MS. By the appropriate selection of ampholyte solutions, whose pH range not necessarily covers the pI values of the analytes, the migration of the components can be controlled, and the impact of the ampholytes on MS detection is decreased. The unique applicability of this setup is shown by testing several parameters, such as the application of volatile electrolyte solutions, the type of the ampholytes, the order and the number of the ampholyte and sample zones. Broad and narrow pH range ampholytes were applied in experiments using uncoated capillaries with different lengths for the analyses of substituted nitrophenol dyes to achieve optimal conditions for the MS detection. Although the sample components are not leaving the pH gradient, due to the decrease in the ampholyte concentration at the position of the components, and because the sample components migrate in charged state, the ionisation is more effective for MS detection.

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“…In contrast to these advantages of cIEF-MS, the major problems in hyphenation are the presence of ampholytes (signal suppression in MS) and the zone broadening from cIEF to MS. Tang et al [2,4] showed that for cIEF-ESI-MS, ampholytes concentrations of more than 0.5% give significant signal suppression and considerably impair sensitivity. However, the number of cIEF-MALDI papers is by far outnumbered by the amount of papers on the coupling of cIEF to ESI (recently reviewed in [5][6][7]). Probably the on-line analysis and higher sampling rate are the reasons for the popularity of ESI over MALDI.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2009

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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.

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Recent developments in capillary isoelectric focusing

Cited by 77 publications

References 143 publications

Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed‐phase liquid chromatography with mass spectrometry for online protein profiling

Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed‐phase liquid chromatography with mass spectrometry for online protein profiling

Sequential injection setup for capillary isoelectric focusing combined with MS detection

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

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