Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF. IV: pH 6–8 intervals

Simó, Carolina; Citterio, Attilio; Righetti, Pier Giorgio

doi:10.1002/elps.200600853

Cited by 21 publications

(18 citation statements)

References 17 publications

(35 reference statements)

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“…Furthermore, to form an almost continuous pH gradient, at least 20 different CAs per pH unit are required [30]. Buffering power, conductivity, mass distribution, and linearity (measured vs. theoretical pH) of ampholyte blends have been investigated before [31][32][33][34][35][36][37]. Therefore, in this paper we will focus on the influence of three different blends of CAs on the detection: UV background absorbance and signal suppression in MALDI.…”

Section: Ampholytesmentioning

confidence: 99%

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Improved repeatability and matrix‐assisted desorption/ionization – time of flight mass spectrometry compatibility in capillary isoelectric focusing

et al. 2008

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Low repeatability of migration time, peak area, and linearity (pI vs. mobilization time) is a problem often encountered in capillary IEF (cIEF) and is mainly caused by protein precipitation and protein-wall interactions. In order to study the influence of these phenomena, the effect of different classes of additives on repeatability of migration time, peak area and linearity of a mixture of seven model proteins has been investigated. Moreover, the influence of these additives on protein signal suppression in MALDI-TOF MS has been studied. The optimal ampholyte blend (stabilizes pH gradient) to be used depends on the selected UV detection wavelength. All tested ampholyte blends show a significant and comparable signal suppression in MS. The best detergent (to prevent precipitation and wall interaction) should be determined for each sample individually, but generally polyethylene oxide and zwitterionic detergents show good repeatability for migration time (RSD <4.5%) and peak area (majority <10%). The RSD of R(2) is <1.3% for the hydrophilic protein mixture. However, these components cause severe signal suppression in MS. Therefore glucoside detergents should preferably be used for MS coupling. Viscosity-increasing agents (for hydrodynamic wall coating and to minimize diffusion) in particular cellulose derivatives, give good repeatability for migration times (RSD <4.5% at lower concentrations), peak area (except for high concentration methylcellulose and hydroxyethylcellulose all within 7.5%), and correlation (pI vs. migration time), but severe signal suppression is observed in MALDI-TOF MS. Overall, cIEF repeatability and linearity can significantly be improved by adding the appropriate components. However, when the system is coupled to a MALDI-TOF MS, compromises have to be made between high repeatability and linearity on one hand and MS signal intensity on the other.

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Section: Ampholytesmentioning

confidence: 99%

“…Since the blends contain different amounts of ampholytes [32,33,37], UV traces at l = 214 nm (a) and 280 nm (b) of Ampholine, HR ampholyte and Pharmalyte, with (black trace) and without (gray trace) the protein mixture (all at a final concentration of 1% w/v). On the x-axis, focusing time (12 min) and mobilization time is plotted.…”

Section: Ampholytesmentioning

confidence: 99%

Improved repeatability and matrix‐assisted desorption/ionization – time of flight mass spectrometry compatibility in capillary isoelectric focusing

et al. 2008

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“…At the end of this long riding along the pH gradients generated by all the commercial brands [20][21][22][23], we can now summarize our data and try to draw educated guesses at what lies ahead. One clear indication emerges: the acidic pH intervals, up to pH ca.…”

Section: New Synthetic Approaches?mentioning

confidence: 99%

“…In a third communication [22], the extreme acidic ranges (typically covering the pH 2.5-4.5 intervals) were explored. In a fourth report, the pH 6-8 intervals were studied [23]. We had to close the cycle by analyzing the most alkaline pH range (pH 9-11) commercially available.…”

Section: Introductionmentioning

confidence: 99%

Mass distribution, polydispersity, and focusing properties of carrier ampholytes for IEF. Part V: pH 9–11 interval

2007

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As a last part of an investigation on all 2-pH-unit intervals of carrier ampholytes (CAs) for IEF (see Electrophoresis 2006, 27, 3919-3934; 2006, 27, 4849-4858; 2007, 28, 715-723) two different lots of Servalyt CAs, in the pH 9-11 range, have been analyzed by a 2-D technique based on preparative Rotofor fractionation followed by capillary electrophoresis mass-spectrometry of 10 out of 20 fractions harvested, in the second dimension. The findings: the two lots contain 65 and 69 different M(r) compounds, in the M(r) interval of 232-667 Da, for a total of 341-387 isoforms, respectively. Since this is a chaotic organic synthesis, the high reproducibility (here demonstrated for the first time during the 40 years of existence of CAs) of the synthetic process (for two batches produced at 6 years of distance) is remarkable, considering that a 94% agreement for the individual chemicals and 88% agreement for the total number of isoforms for the two lots is found. It is additionally demonstrated that the lower pI species are accompanied by considerably more isoforms than the high pI forms and that in all cases such isoforms consist of family of compounds clustered around the pI of the parental form, with a pI spread of ca. 0.1-0.2 pH units.

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“…CAs are usually used as a very heterogeneous mixture in isoelectric focusing (IEF). In such a mixture, covering, for example a pH 3-10 range, a very high number of compounds can be found [9][10][11]. To obtain solutions containing only CAs with close isoelectric points (pI), we fractionated a wide pH range mixture of CAs by preparative IEF in a granulated gel [12].…”

Section: Introductionmentioning

confidence: 99%

Integration of various stacking processes in carrier ampholyte-based capillary electrophoresis

Busnel

Saux

Descroix

et al. 2008

Journal of Chromatography A

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Field-enhanced sample stacking, field-enhanced sample injection as well as electrokinetic supercharging have been successfully integrated in carrier ampholyte-based capillary electrophoresis. Through the analysis of different test sample mixtures, it has been shown that the carrier ampholyte-based background electrolytes, in spite of their very low conductivity, allow efficient online preconcentration of analytes by fieldamplified techniques. Sensitivity enhancement factors of the same magnitude as those usually encountered with classical conductive background electrolytes have been obtained in such carrier ampholyte-based buffers. Depending on the online preconcentration method that has been integrated, sensitivity enhancement factors between 50 and several thousands have been reached.

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Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF. IV: pH 6–8 intervals

Cited by 21 publications

References 17 publications

Improved repeatability and matrix‐assisted desorption/ionization – time of flight mass spectrometry compatibility in capillary isoelectric focusing

Improved repeatability and matrix‐assisted desorption/ionization – time of flight mass spectrometry compatibility in capillary isoelectric focusing

Mass distribution, polydispersity, and focusing properties of carrier ampholytes for IEF. Part V: pH 9–11 interval

Integration of various stacking processes in carrier ampholyte-based capillary electrophoresis

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