Capillary isoelectric focusing as a tool in the examination of antibodies, peptides and proteins of pharmaceutical interest

Liu, X.; Sosic, Zoran; Krull, Ira S.

doi:10.1016/0021-9673(95)01357-1

Cited by 77 publications

(30 citation statements)

References 50 publications

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“…C apillary isoelectric focus ing (clEF) carried ou t in fused silica capillaries was orig inally demonstrated by Hjerten and Zhu over ten years ago [1]. It has subsequently found widespread use in the separation and analysis of polypeptide and protein mixtures [2][3][4]. The two major advantages of clEF over conventional slab gel electrophoresis are the enhanced separation efficiencies as well as a substantial reduction in Joule heating [5].…”

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confidence: 99%

On-line desalting of physiologically derived fluids in conjunction with capillary isoelectric focusing-mass spectrometry

Clarke

Tomlinson

Naylor

1997

J. Am. Soc. Mass Spectrom.

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Capillary isoelectric focusin g (clEF) coupled to mass spectrometry (cIEF-MS) offers a potentially very powerful analytical tool for the investigation of physiological samples. The high resolvin g capabilities of clEF in combination with the high sensitivity and enhanced structural information provided by MS is highly desirable for the analysis of complex samples. How ever a major limitation of the technique has alw ays been the requ irement to desalt samples pr ior to clEF analysis. Such desalting normally occurs off-line and therefore adds complexity and the possibility of sample loss or contamina tion. In this study we demonstrate the use of a modified clEF protocol which enables samples containing ph ysiological levels of salts to be de salted on-line, within the clEF capillary. This new technique is very fast and efficient, allowi ng the direct analysis of a ph ysiologically derived fluid that contains a complex mix of proteins, such as human cerebrospinal fluid by clEF-MS in a single step experiment. C apillary isoelectric focus ing (clEF) carried ou t in fused silica capillaries was orig inally demonstrated by Hjerten and Zhu over ten years ago [1]. It has subsequently found widespread use in the separation and analysis of polypeptide and protein mixtures [2-4]. The two major advantages of clEF over conventional slab gel electrophoresis are the enhanced separation efficiencies as well as a substantial reduction in Joule heating [5]. Experimentally, the clEF proc ess consists of an initi al focusing step that is based on the isoelectric point (pl) values of ind ividual anal ytes. It is subseque ntly followed by an analyte mobilization stage affected either by electrophoresi s or pressure [6]. This is shown schema tically in Figure 1. Capillary IEF has been used exten sively with conv entional UV detection; however, the use of clEF coupled to mass spe ctrometry (clEF-MS) has to date received scant attention [7,8). The us e of clEF-MS affords a highl y efficient sepa ration technique with the capability to stru cturally characterize anal ytes deri ved from complex mixtures. This is Add ress repri nt requ ests to Stephen Nay lor, Biomedical Mass Spectrometry Facility,

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confidence: 99%

On-line desalting of physiologically derived fluids in conjunction with capillary isoelectric focusing-mass spectrometry

Clarke

Tomlinson

Naylor

1997

J. Am. Soc. Mass Spectrom.

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“…It has subsequently been widely utilized in the analysis of peptides and proteins (Liu et al, 1996;Pritchett, 1996;Wu and Pawliszyn, 1994;Rodríguez-Díaz et al, 1997;Righetti et al, 1996;Shen et al, 1999;Shen et al, 2000;Lupi et al, 2000;Hu et al, 2001). The use of a fused silica capillary as the support for the electrophoretic matrix, rather than a traditional slab gel, remedies many of the problems inherent in gel electrophoretic techniques.…”

Section: Introductionmentioning

confidence: 97%

Capillary isoelectric focusing–mass spectrometry: analysis of protein mixtures from human body fluids

Clarke

Naylor

2002

Biomedical Chromatography

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Isoelectric focusing within a fused silica capillary (cIEF) has proved to be a powerful and practical method for high-resolution separation of analytes from complex biological mixtures. This technique overcomes many of the problems of isoelectric focusing within slab gel media. However current cIEF systems commonly utilize UV detection which limits the detail of analyte structural information that is obtained during analysis. The use of mass spectrometry (MS) as the detection system provides much greater structural information about the detected analytes allowing accurate relative molecular mass (M(r)) determination for proteins and polypeptides. We have constructed a cIEF-MS interface and compared the separation of standard proteins analyzed by cIEF-UV with cIEF-MS. This allowed rapid optimization of the cIEF-MS system performance. Further we have demonstrated the use of MS as a detection system provides accurate M(r) information and can provide analyte modification details. These factors increase the likelihood of absolute identification for physiological proteins within complex in vivo-derived mixtures. To demonstrate the value of cIEF-MS in such analyses we have undertaken an examination of cerebrospinal fluid (CSF), and tentatively identified a number of constituent proteins. We have also analyzed whole blood from control and diabetic patients. We show that glycated alpha- and beta- chains of hemoglobin are found in almost equal abundance in diabetic patient blood. From these results we suggest cIEF-MS is an efficient and useful tool for the separation and examination of in vivo-derived analytes within physiological fluids.

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“…The examples of separation of new pI markers together with derivatized proteins by capillary IEF with photometric or fluorometric detection by Horka was presented [7]. Most publications using cIEF concerns proteomics and biopharmaceutical applications [8][9][10][11][12][13].…”

Section: Introductionmentioning

confidence: 99%

PI-Based Separation of Tissue Proteins Realized with the Use of Two-Dimensional Gel Electrophoresis or Alternatively Capillary Isoelectric Focusing

Olędzka¹

2012

TOPROTJ

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Abstract:A two-dimensional gel electrophoresis (2D-GE) and capillary isoelectric focusing (cIEF) were performed subsequently to analyze protein mixtures from laboratory animal tissues (liver, muscle, and mammary gland). Both techniques were compared and evaluated. It was found that cIEF can be a valuable alternative in comparison to 2D-GE. Especially considering the goals associated with the determination of far the separated proteins, cIEF possesses perspective analytical advantages.

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Capillary isoelectric focusing as a tool in the examination of antibodies, peptides and proteins of pharmaceutical interest

Cited by 77 publications

References 50 publications

On-line desalting of physiologically derived fluids in conjunction with capillary isoelectric focusing-mass spectrometry

On-line desalting of physiologically derived fluids in conjunction with capillary isoelectric focusing-mass spectrometry

Capillary isoelectric focusing–mass spectrometry: analysis of protein mixtures from human body fluids

PI-Based Separation of Tissue Proteins Realized with the Use of Two-Dimensional Gel Electrophoresis or Alternatively Capillary Isoelectric Focusing

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