Chromatofocusing

Frey, Douglas D.; Narahari, Chittoor R.; Bates, Ronald C

doi:10.1038/npg.els.0002681

Cited by 3 publications

(2 citation statements)

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“…6,7) ere are many LC separation modes applied to the frontend of MS. For example, chromatofocusing is one of useful separation modes that allows separation of proteins based on their isoelectric point (pI). 8) Proteins which have di erent pI migrate at di erent speed through the column, and combining this technique with MS can be powerful tool for identifying unknown analytes because it is possible to obtain pI value and structural information at the same time. Octadecyl silica (ODS) is the most commonly used mode in LC-MS.…”

Section: Introductionmentioning

confidence: 99%

Salt Tolerance Enhancement of Liquid Chromatography-Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Using Matrix Additive Methylenediphosphonic Acid

et al. 2014

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Mass spectrometry (MS) is a highly sensitive analytical technique that is o en coupled with liquid chromatography (LC). However, some bu ering salts used in LC (e.g., phosphate and tris(hydroxymethyl) aminomethane (Tris)) are incompatible with MS since they cause ion-source contamination and signal suppression. In this study, we examined salt tolerance of MALDI and applied a matrix additive methylenediphosphonic acid (MDPNA) to reduce salt-induced signal suppression. MDPNA signi cantly improved the salt tolerance of MALDI-MS. Using ammonium formate bu er at pH 5.0, the e ective range of bu ering salt concentration in MALDI-MS using MDPNA was estimated up to 250 mM. MDPNA reduced signal suppression caused by bu ering salts at pH 4.0 to 8.0. We observed that MDPNA e ectively worked over a wide range of bu er conditions. MDPNA was further applied to hydrophilic interaction chromatography (HILIC) and chromatofocusing-MALDI-MS. As a result, the analytes in the eluent containing high-concentration salts were detected with high sensitivity. us, our study provides simple and fast LC-MALDI-MS analysis technique not having strict limitation of bu ering condition in LC by using matrix additive MDPNA.

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Section: Introductionmentioning

confidence: 99%

Salt Tolerance Enhancement of Liquid Chromatography-Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Using Matrix Additive Methylenediphosphonic Acid

et al. 2014

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“…Efforts have therefore been made to identify ways to replace polyampholytes with mixtures of simple monoprotic and diprotic buffers. Frey and coworkers have made among the most significant contributions (Frey, ; Frey et al, ; Frey and Strong, ; Kang and Frey, ; Kang et al, ), but others have made important contributions as well (Liu and Anderson, , , ). In addition, although their work did not focus on chromatofocusing, Yang et al (), Tsonev and Hirsh () and Xu and Glatz () have conducted extensive studies mapping the pH and pI dependence of protein behavior in ion‐exchange systems.…”

Section: Introductionmentioning

confidence: 99%

Improved isoelectric focusing chromatography on strong anion exchange media via a new model that custom designs mobile phases using simple buffers

Creagh

Haynes

2013

Biotech & Bioengineering

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Isoelectric chromatofocusing (ICF), a mode of chromatography by which proteins are separated based on changes in their charge state with pH, is widely used at analytical scales and finding increasing interest in biologics manufacturing due to its exceptional resolving power. Here, a method is described for using simple monoprotic and diprotic buffers to create stable mobile phases for sample loading on a strong anion exchange column and for achieving an elution pH gradient of desired shape covering any pH range from pH 10.0 to 3. The buffers used are selected to satisfy cost constraints, and to permit facile detection of eluted biologics by UV spectroscopy and mass spectrometry. The method exploits a new model described here that combines multiple-chemical and adsorption-equilibria theory to enable in silico tailoring of elution pH profiles using mixtures of these simple buffers. It is shown to provide a versatile platform for optimizing and conducting ICF of protein mixtures on strong anion exchange media.

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Reducing Sample Complexity in Proteomics by Chromatofocusing with Simple Buffer Mixtures

Shen

Bieberich

et al. 2008

Methods in Molecular Biology™

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Chromatofocusing has many potential applications in the field of proteomics, such as for the isolation and removal of major sample components to facilitate the analysis of low-abundance components, and for sample prefractionation prior to a subsequent separation using SDS-PAGE, narrow-pI-range 2D-PAGE, or additional chromatography steps. However, the chromatofocusing techniques that are most commonly used employ propriety polyampholyte elution buffers and highly specialized column packings, both of which limit the use of chromatofocusing in practice. To expand the range of application for this technique, this chapter considers chromatofocusing methods which employ common ion-exchange column packings and elution buffers which are simple mixtures of readily available buffering species. Of particular interest is the use of chromatofocusing with a multistep pH gradient for the fractionation of protein mixtures into narrow-pI-range fractions. The cross-contamination characteristics of these fractions using SDS-PAGE are also assessed.

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Chromatofocusing

Abstract: Chromatofocusing is a form of gradient elution chromatography performed using an ion‐exchange column packing and an internally generated pH gradient that travels through the column as a retained pH front.

Cited by 3 publications

References 11 publications

Salt Tolerance Enhancement of Liquid Chromatography-Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Using Matrix Additive Methylenediphosphonic Acid

Salt Tolerance Enhancement of Liquid Chromatography-Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Using Matrix Additive Methylenediphosphonic Acid

Improved isoelectric focusing chromatography on strong anion exchange media via a new model that custom designs mobile phases using simple buffers

Reducing Sample Complexity in Proteomics by Chromatofocusing with Simple Buffer Mixtures

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