Effects of the sample matrix on the separation of peptides by high performance capillary electrophoresis

Satow, T.; Machida, Akiko; Funakushi, Kayo; Palrnieri, Richard

doi:10.1002/jhrc.1240140415

Cited by 18 publications

(8 citation statements)

References 3 publications

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“…The two most common factors, which cause poor resolution in 2‐D gels, are the amount of salt in the sample and the extent of denaturation of proteins. CE separation is also adversely affected by salt concentration 32 and nondenatured proteins. This observation was confirmed by preparing the protein samples in three different buffers (2‐DE sample buffer, 2‐DE sample buffer containing 50 mM NaCl, and 2‐DE sample buffer without denaturants; urea and thiourea) to reproduce low‐quality 2‐DE protein samples.…”

Section: Resultsmentioning

confidence: 99%

CE‐based sample quality assessment prior to 2‐D gel electrophoresis: Towards the standardization of gel‐based proteomics

Techanukul

Pereira

Lipka

et al. 2010

J of Separation Science

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2-DE remains one of the most commonly used separation techniques for complex protein mixtures. This article describes a new approach to 2-DE sample assessment using SDS capillary gel electrophoresis (in Beckman Coulter sieving medium) combined with multi-pixel detection. The performance of this platform was investigated using protein samples prepared for 2-DE. The capability to characterize 2-DE sample was tested and the results show that the repeatability of peak migration time and intensity are better than 2% RSD. The system gives good resolution, accurate molecular mass assignment, as well as absolute and relative quantification of proteins. Notably, this study also demonstrates the use of this platform to screen the quality of simple and complex 2-DE samples. Implementation of this technique in the proteomics workflow will not only improve the success rate of 2-DE, but will also enable sample verification before 2-DE and allow the relative quantification of proteins. The validation of differential protein expression is also demonstrated using the combined information of relative molecular mass and relative quantification. It is the first time that a rapid and visual evaluation method is reported for the quality assessment of 2-DE samples.

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Section: Resultsmentioning

confidence: 99%

CE‐based sample quality assessment prior to 2‐D gel electrophoresis: Towards the standardization of gel‐based proteomics

Techanukul

Pereira

Lipka

et al. 2010

J of Separation Science

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“…The increased electrmsmotic flow strongly suggests a timedependent and buffer concentration-dependent interaction between the phosphate buffer and silica surface (12,13). In addition, the relatively high ionic strength allows for a greater variation in sample matrix conditions yet still promotes conditions favorable for focussing or sample stacking (14). Short capillaries are advantageous for protein separations as their residence times are generally short, leading to a decreased chance of adsorption.…”

Section: Utility Of Short Smll Id Capillariesmentioning

confidence: 98%

“…Although the capillary length was increased (57 cm to 107 cm) and the voltage was reduced (30 kV to 20 kV), the results from the micropreparative run ( Figure 6A) compare favorably to the analytical run. The high ionic strength buffer promotes sample focusing for the relatively high concentrations of protein and buffer used during the tryptic digestion (14). This effect along with the increase in resolution more than compensates for the longer run times and increased Joule heat experienced with the higher ionic strength.…”

Section: Effect Of Capillary Length I Addition Of Zwitterionsmentioning

confidence: 99%

Use of High Ionic Strength Buffers for the Separation of Proteins and Peptides with Capillary Electrophoresis

Chen¹,

Kelly²,

Palmieri³

et al. 1992

Journal of Liquid Chromatography

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Journal of Liquid ChromatographyPublication details, including instructions for authors and subscription information: ABSTRACTThe use of high ionic strength buffers with capillary electrophoresis in untreated fused silica capillaries is demonstrated for the separation of proteins and peptides. Short, small i.d. (20-25 pm) capillaries are useful as a quick screening tool for protein analysis. Electropherograms of standard proteins run from pH 5.0 to 10.0 in 0.5 M sodium phosphate buffers suggest that at these high ionic strengths, the proteins (relative to the electrmmotic flow markers) are not always predictable functions of their PI'S. Consecutive runs of standard proteins yield excellent migration time and peak area reproducibility. Milk proteins, particularly the caseins, could be separated with the addition of 4 M urea to the buffer. Alternatively, the addition of a zwitterion to a phosphate-based buffer provide different selectivities for the protein standards. The accompanying reduction in ionic strength allows for

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“…The procedure described above results in the injection and detection of all anions in a sample, not just (P i ) n 67. Analysis of (P i ) n by indirect detection works best on samples that are free of salts besides (P i ) n .…”

Section: Experimental Designmentioning

confidence: 99%

Analysis of Inorganic Polyphosphates by Capillary Gel Electrophoresis

Lee

Whitesides

2010

Anal. Chem.

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This paper describes the development of a method that uses Capillary Gel Electrophoresis (CGE) to analyze mixtures of inorganic polyphosphate ((Pi)n). Resolution of (Pi)n on the basis of n, the number of residues of dehydrated phosphate, is accomplished by CGE using capillaries filled with solutions of poly(N,N-dimethylacrylamide) (PDMA) and indirect detection by the UV-absorbance of a chromophore, terephthalate, added to the running buffer. The method is capable of resolving peaks representing (Pi)n with n up to ~70; preparation and use of authentic standards enables the identification of peaks for (Pi)n with n = 1 - 10. The main advantages of this method over previously reported methods for analyzing mixtures of (Pi)n (e.g., gel electrophoresis, CGE using polyacrylamide-filled capillaries) are its resolution, convenience, and reproducibility; gel-filled capillaries are easily regenerated by pumping in fresh, low-viscosity solutions of PDMA. The resolution is comparable to that of ion-exchange chromatography and detection of (Pi)n by suppressed conductivity. The method is useful for analyzing (Pi)n generated by the dehydration of Pi at low temperature (125 - 140 °C) with urea, in a reaction that may have been important in prebiotic chemistry. The method should also be useful for characterizing mixtures of other anionic, oligomeric or polymeric species without an intrinsic chromophore (e.g., sulfated polysaccharides, oligomeric phospho-diesters).

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Effects of the sample matrix on the separation of peptides by high performance capillary electrophoresis

Cited by 18 publications

References 3 publications

CE‐based sample quality assessment prior to 2‐D gel electrophoresis: Towards the standardization of gel‐based proteomics

CE‐based sample quality assessment prior to 2‐D gel electrophoresis: Towards the standardization of gel‐based proteomics

Use of High Ionic Strength Buffers for the Separation of Proteins and Peptides with Capillary Electrophoresis

Analysis of Inorganic Polyphosphates by Capillary Gel Electrophoresis

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