Combining capillary electrophoresis with mass spectrometry for applications in proteomics

Simpson, David C.; Smith, Richard D.

doi:10.1002/elps.200410132

Cited by 177 publications

(142 citation statements)

References 164 publications

(145 reference statements)

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“…The use of a hydroxypropylmethylcellulose-coated column significantly improved the separation of basic histones. A simple dialysis step prior to CE separation and the use of coaxial sheath liquid flow between the CE and ESI-MS interface made the online CE-ESI-MS possible [108][109][110][111]. Despite the potential power of the approach, CE-MS of histones is difficult due to:…”

Section: Hp Capillary Electrophoresis-msmentioning

confidence: 99%

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Ren

Freitas

2007

Expert Review of Proteomics

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Due to the intimate interactions between histones and DNA, the characterization of histones has become the focus of great attention. A series of mass spectrometry-based technologies have been dedicated to the characterization and quantitation of different histone forms. This review focuses on the discussion of mass spectrometry-based strategies used for the characterization of histones and their post-translational modifications. Keywordshistone; mass spectrometry; post-translational modification Histones are a group of highly conserved proteins throughout eukaryotic evolution [1]. The core histones (H4, H3, H2A and H2B) form an octamer, in which a H2A-H2B dimer associates on each side of the H3-H4 tetramer through an interaction between the C-terminal halves of H2B and H4 [2]. The negatively charged DNA superhelix wraps around the histone octamer to form repeating nucleosome cores, which further assemble into higher order chromatin fibers stabilized by the linker histones (H1 and H5) and linker DNA [3]. The histone fold regions in core histones are highly conserved α-helices, which are connected by two loops. The third histone structural motif comprises the flexible and irregular histone tails, which are extended and reach outside of the nucleosomal unit to interact with DNA [4]. Challenge of molecular characterization: post-translational modifications & sequence variationsAs a building block of the nucleosome, histones play an important role in chromatin assembly and affect the interactions between DNA and other chromatin-associated proteins. Histones regulate gene transcription through their diverse post-translational modifications (PTMs), which include lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, lysine ubiquitination, lysine sumoylation, and poly-ADP-ribosylation of glutamic acid [5][6][7][8]. Among all the PTMs, histone acetylation and methylation have been most †

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Section: Hp Capillary Electrophoresis-msmentioning

confidence: 99%

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Ren

Freitas

2007

Expert Review of Proteomics

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“…Soft ionization techniques such as MALDI and ESI allowed a combination of CE and MS [99][100][101][102][103]. In CE-MS the proteins separated by CE are typically introduced into MS by an electrospray [104].…”

Section: Msmentioning

confidence: 99%

“…CZE provided data comparable to gel electrophoresis for analysis of serum proteins [187][188][189][190][191]. Combination of CZE with MS resulted in additional data for discovery of biomarkers and better medical diagnostics [100,192]. Through the analysis of serum proteins, CZE could identify inflammation, nephrotic syndrome, bisalbuminemia, and a 1 -antitrypsin deficiency [193].…”

Section: Proteins In Bloodmentioning

confidence: 99%

Capillary electrophoresis of proteins 2003–2005

Dolnı́k¹

2006

Electrophoresis

139

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This review article with 304 references describes recent developments in CE of proteins, and covers the two years since the previous review (Hutterer, K., Dolník, V., Electrophoresis 2003, 24, 3998-4012) through Spring 2005. It covers topics related to CE of proteins, including modeling of the electrophoretic migration of proteins, sample pretreatment, wall coatings, improving separation, various forms of detection, special electrophoretic techniques such as affinity CE, CIEF, and applications of CE to the analysis of proteins in real-world samples including human body fluids, food and agricultural samples, protein pharmaceuticals, and recombinant protein preparations.

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“…It has been successfully employed in the characterization of the proteome of E. Coli with the detection of 400 to 1000 proteins [9,10]. This technique requires the use of low concentrations, which compromises peak width, or complete removal of the carrier ampholytes with a microdialysis setup, to obtain an on-line MS signal [11,12]. Problems with this technique are protein adsorption to the capillary walls, which lead to reproducibility problems [13] and the effect of protein concentration on the pH gradient [14].…”

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confidence: 99%

Gradient chromatofocusing-mass spectrometry: A new technique in protein analysis

Lian

Hribar

Zhou

et al. 2008

J. Am. Soc. Mass Spectrom.

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A new analytical technique, gradient chromatofocusing-mass spectrometry (gCF-MS), was developed employing ion-exchange high-performance liquid chromatography (HPLC) interfaced to an electrospray-quadrupole mass spectrometer in the determination of proteins. There have been few reports, if any, of a HPLC-MS technique for proteins in which the ion-exchange column is directly interfaced to the mass spectrometer. The employment of a linear pH gradient elution scheme directly interfaced to mass spectrometry is also unique in the present work. The technique was demonstrated by the separation of six proteins (carbonic anhydrase II, enolase, ␤-lactoglobulin A, lactoglobulin B, soybean trypsin inhibitor, and amyloglucosidase) employing a descending linear pH gradient from pH 9 to 2.6 on a 50 mm ϫ 2.1 mm DEAE HPLC column using volatile buffer components. A signal enhancement solution consisting of 8% formic acid in acetonitrile was pumped post-column and was mixed 1:1 with column effluent and then directed on-line into the mass spectrometer. Molecular masses of the proteins were determined within Ϯ0.010% to 0.033% (Ϯ100 to 330 ppm) with peak height total ion current detection limits of 4 to 78 pmol of injected amounts (S/N ϭ 3). This technique is applicable to the analysis of proteins and other charged molecules. T here is great interest in developing a mass spectrometry (MS)-compatible high-performance liquid chromatography (HPLC) technology which performs charge-based separations, a principal dimension in the 2D-gel electrophoresis technique, for application in proteomic and protein characterization studies. Development of such a HPLC technique would be progress towards addressing the limitations of the 2D-gel electrophoresis technique, which has been hampered by its design incompatibility for direct interfacing with MS (requiring excision of each protein band from the gel), limitation in quantitative dynamic range, inability of determining small proteins (5-8000 Da), high labor intensity, long sample run times, as well as other disadvantages [1,2].Development of charged-based HPLC techniques directly coupled to mass spectrometry (MS) has been a difficult challenge. This is because the mobile phase salts commonly used in ion-exchange chromatography, the most used charge-based HPLC technique, suppress the MS signal. The most utilized approach incorporating ion-exchange chromatography with mass spectrometry has been to use a 2D ion-exchange/reversed-phase HPLC technique, directing the ion-exchange fractions to a reversed-phase column for further separation and removal of salt before on-line mass spectrometry. This has been applied to proteomic studies in the analysis of proteins [3] and protein digests [4,5]. A "stair step" gradient is employed for the ion-exchange dimension. This first dimension thus serves as a crude initial separation step, and is not the stage that is directly interfaced to the mass spectrometer.The application of two-dimensional chromatography using the combination of chromatofocusing and reversed-p...

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Combining capillary electrophoresis with mass spectrometry for applications in proteomics

Cited by 177 publications

References 164 publications

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Capillary electrophoresis of proteins 2003–2005

Gradient chromatofocusing-mass spectrometry: A new technique in protein analysis

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