Capillary electrophoresis–mass spectrometry for the analysis of intact proteins

Haselberg, Rob; Jong, Gerhardus J. de; Somsen, Govert W.

doi:10.1016/j.chroma.2007.05.048

Cited by 169 publications

(135 citation statements)

References 197 publications

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“…A single analytical technique for the characterization of biologicals is generally not sufficient [54,55].…”

Section: Direct Analytical Characterization Of the Productsmentioning

confidence: 99%

“…8.0: 01/2008:0951) [58]. In CE, the separation is based on charge differences [59], in our case a loss of one positive charge of lysine with the simultaneous addition of negative charges of NOTA, whereas in RP HPLC mainly the decrease in hydrophobicity due to the attachment of NOTA leads to the separation [56]. Overall, the 1:1 and 3:1 samples show similar degrees of somatropin modification as measured with CE-MS compared to LC-MS (Fig.…”

Section: Direct Analytical Characterization Of the Productsmentioning

confidence: 99%

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Analytical characterization of NOTA-modified somatropins

Bracke

Wynendaele

D׳Hondt

et al. 2014

Journal of Pharmaceutical and Biomedical Analysis

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Highlights• NOTA-labeled somatropins were synthesized using different NOTA:protein ratios.• Direct LC-MS and CE-MS approaches indicated multiple substitution degrees.• Bottom-up approaches gave structural insights and information on the labeling yield.• Lys-70 (in silico calculated pKa of 8.3) is the NOTA-modification hotspot.• We report a synthesis procedure for the production of target-specific radiopharmaceuticals.

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“…A single analytical technique for the characterization of biologicals is generally not sufficient [54,55].…”

Section: Direct Analytical Characterization Of the Productsmentioning

confidence: 99%

Section: Direct Analytical Characterization Of the Productsmentioning

confidence: 99%

Analytical characterization of NOTA-modified somatropins

Bracke

Wynendaele

D׳Hondt

et al. 2014

Journal of Pharmaceutical and Biomedical Analysis

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“…[10] CE-MS was introduced about two decades ago and has found different and increasingly accepted applications. [11,12] The high resolution and efficiency of CE allied to the universality and sensitivity of MS are invaluable when compared with those of other detection systems such as UV-Vis spectrophotometry and laser-induced fluorescence (LIF), which provide little information about the analyte's structure. [13,14] For instance, the use of MS associated with computational tools is highly recommended in studies of complex samples such as proteins, peptides, and metabolites because information such as molecular mass and structure (tandem mass spectrometry) is indispensable for the characterization and quantification of these analytes.…”

Section: Introductionmentioning

confidence: 99%

“…[1,2,3,4] CE provides high resolution and efficiency, which are very important parameters in separation processes. Another advantage of CE is its low operational cost, even with organic solvents because it uses negligible volumes compared with the amounts used in HPLC, even in capillary-scale applications.…”

Section: Introductionmentioning

confidence: 99%

Homemade Capillary Electrophoresis Coupled to a Mass Spectrometer

Moraes

Carrilho

Assunção

2014

Journal of Liquid Chromatography & Related Technologies

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A system was developed in our laboratory to couple capillary electrophoresis with mass spectrometry. The capillary electrophoresis system was equipped with a high voltage supply, and a microcontroller with assembly language programming was developed for the computational control of the system. The MS system was a commercial Thermo Finningan LCQ ion trap mass spectrometer. The robustness of the coupled system was evaluated using standard protein samples (lysozyme, aprotinin, and bovine albumin) and tryptic digests of lysozyme. The system showed positive results in terms of robustness, allowing for the separation of digested proteins and the identification of 33% of the total amino acids in a protein (6 of the 18 expected peptides). The limit of detection was in the order of 1 picomole (signal-to-noise ratio), which was considered satisfactory for this system. The system shows high versatility in tandem coupling and combinations with other analytical procedures.

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“…CE is one of the techniques of choice for separation of the protein isoforms that are a result of microheterogeneity arising in the biosynthesis of a large number of proteins [1][2][3][4]. In CE, protein isoforms (e.g.…”

Section: Introductionmentioning

confidence: 99%

A multiway approach for classification and characterization of rabbit liver apothioneins by CE‐ESI‐MS

et al. 2008

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We applied a multiway approach to extract information from the analysis of protein isoforms by CE-ESI-MS. Metallothioneins (MT) are low-molecular-weight proteins (6-7 kDa) with a strong affinity for heavy-metal ions. Rabbit liver MT-I and MT-II fractions are purified from MT samples. At low pH, the bound metal ions were released from the amino acid structures, giving rise to apothioneins. MT-I, MT-II and MT apothioneins, which are complex mixtures of protein isoforms, were analyzed by CE-ESI-MS. After data pre-processing, parallel factor analysis (PARAFAC) and multivariate curve resolution-alternating least squares (MCR-ALS) were applied to the data sets. In both cases, the models enabled classification of the protein samples and identification of their characteristic sub-isoforms using a set of three components. MCR-ALS required an initial estimate of the pure mass spectra of the three components. Thus, PARAFAC loadings were used to initialize the MCR-ALS optimization. The classifications obtained with MCR-ALS were slightly better than those obtained with PARAFAC, probably because MCR-ALS was less affected by the small migration time shifts of the preprocessed electropherograms. However, no differences were found between the pure mass spectra of the three components in either model. Finally, MCR-ALS allowed us to obtain an individual electrophoretic profile of each of the three components for each of the samples analyzed, which proved valuable for characterization and quantification purposes.

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Capillary electrophoresis–mass spectrometry for the analysis of intact proteins

Cited by 169 publications

References 197 publications

Analytical characterization of NOTA-modified somatropins

Analytical characterization of NOTA-modified somatropins

Homemade Capillary Electrophoresis Coupled to a Mass Spectrometer

A multiway approach for classification and characterization of rabbit liver apothioneins by CE‐ESI‐MS

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