Combination of COFRADIC and high temperature – extended column length conventional liquid chromatography: A very efficient way to tackle complex protein samples, such as serum

Sandra, Koen; Verleysen, Katleen; Labeur, Christine; Vanneste, Lies; D'hondt, Filip; Thomas, Grace; Kas, Koen; Gevaert, Kris; Vandekerckhove, Joël; Sandra, Pat

doi:10.1002/jssc.200600425

Cited by 16 publications

(17 citation statements)

References 30 publications

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“…In a series of reports, we described a highly efficient 1D-LC system consisting of a series of conventional columns operated at elevated temperature [76][77][78] for the analysis of complex protein samples. One particular essay described the extension of the COFRA-DIC procedure (COmbined FRActional DIagonal Chromatography) to a column length of 1 m [76].…”

Section: High Efficiency Lc In the First Dimensionmentioning

confidence: 99%

Tryptic digest analysis by comprehensive reversed phase×two reversed phase liquid chromatography (RP‐LC×2RP‐LC) at different pH's

François

Cabooter

Sandra

et al. 2009

J of Separation Science

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As an alternative to the classical approach of combining strong cation exchange SCX-LC and RP-LC for the separation of complex proteomic samples, this essay describes the online comprehensive RP-LCxRP-LC separation of BSA and human blood serum. High orthogonality and peak capacity are achieved through the application of a significantly different pH in the two dimensions. The coupling of fused-core columns in series ensures high efficiency in the first dimension, while a previously designed interface with parallel second dimension columns further enhances the separation capability of the comprehensive system.

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Section: High Efficiency Lc In the First Dimensionmentioning

confidence: 99%

Tryptic digest analysis by comprehensive reversed phase×two reversed phase liquid chromatography (RP‐LC×2RP‐LC) at different pH's

François

Cabooter

Sandra

et al. 2009

J of Separation Science

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“…Thus, one COFRADIC replicate used approximately 3.88 GB. For technical details about the COFRADIC methodology and the mass spectrometer settings used to generate the data, we refer the reader to the paper of Sandra et al [2] Methods A typical feature of COFRADIC data, or more generally LC-MS data, is that a peptide signal s(t, m/z) is present in two dimensions, whereLoad new COFRADIC experiment.…”

Section: Cofradic Mass Spectramentioning

confidence: 99%

“…In these situations one can consider using three peaks, while setting the height of the fourth (undetected) peak to zero. Note that this is a conservative approach, as the fourth isotopic variant will contribute to the Pearson's χ 2 error in Eqn (2). This can be interpreted as a penalization for not finding the fourth peak.…”

Section: Peptide Peak Validationmentioning

confidence: 99%

“…To provide a better understanding of the complexity of the data generated from the COFRADIC methodology, the procedure and typical characteristics of this separation technique will be briefly explained. For more details on the COFRADIC methodology and the MS settings, we refer to the paper of Sandra et al, [2] which describes the procedures quite extensively.…”

Section: Introductionmentioning

confidence: 99%

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A strategy for the prior processing of high‐resolution mass spectral data obtained from high‐dimensional combined fractional diagonal chromatography

Valkenborg

Thomas²,

Krols³

et al. 2008

J. Mass Spectrom.

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Combined fractional diagonal chromatography (COFRADIC) is a novel suite of gel-free technologies for the identification of biomarkers in complex peptide mixtures. For this purpose, reversed-phase high performance liquid chromatography (HPLC) technology and, in this case, matrix assisted laser desorption /ionization-time of flight (MALDI-TOF) mass spectrometers are extensively used. The particular characteristic of COFRADIC mass spectrometry data is the high number of chromatographic fractions, over which a peptide can be scattered. This can obstruct the quantification of the peptide abundance in the biological sample, which is required for statistical analysis. On the other hand, because of the superior peptide sorting properties of the methodology, the mass spectra become less crowded. Consequently, each peptide appears in a mass spectrum as a series of peaks with peak heights proportional to the probability of occurrence of the isotopic variants of the peptide. In this manuscript, we propose an analysis strategy concerned with the preprocessing of COFRADIC mass spectra prior to a downstream statistical analysis. The preprocessing algorithm produces for each mass spectrum a peptide list by exploiting the characteristic features that should be associated with peaks corresponding to an isotopically resolved cluster of peptide peaks. This reduction step is necessary to facilitate the clustering used in a next step to assemble the validated monoisotopic peptide peaks found over several fractions into a single peptide abundance. To assess the performance of the algorithm, two technical experiments were conducted. The proposed strategy is memory and computationally efficient.

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“…The only additional requirement is an appropriate mobile phase and column heating system [20,[22][23][24]. The use of elevated column temperatures in RP-LC to achieve high separation efficiencies have been investigated extensively [5,[25][26][27][28][29][30] …”

mentioning

confidence: 99%

High-efficiency hydrophilic interaction chromatography by coupling 25cm×4.6mm ID×5μm silica columns and operation at 80°C

Louw

Lynen

Hanna-Brown

et al. 2010

Journal of Chromatography A

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Recently, hydrophilic interaction chromatography (HILIC) has emerged as a valuable orthogonal tool to reversed-phase liquid chromatography (RP-LC) as it allows for resolution of highly polar ionisable compounds. The relationships between separation efficiency, column length and speed of analysis for 4.6 mm ID x 5 µm silica particle columns in HILIC are demonstrated using kinetic plots. The kinetic plots constructed for conventional pressure systems operating at 350 bar and at 30 o C and 80°C are confirmed using experimental data for different column lengths. Efficiencies of more than 130,000 theoretical plates could be achieved by connecting up to six columns of 25 cm. As expected, a significant gain in analysis speed without loss of efficiency could be obtained by operating at 80°C compared to 30°C. The advantages of using long columns in HILIC in combination with elevated column temperature for the pharmaceutical industry are illustrated using test mixtures comprised of commercially available ionisable compounds (including some containing functional groups with potential genotoxic typical structural alerts) as well as real polar ionisable pharmaceuticals. Keywords:Hydrophilic interaction liquid chromatography, high efficiency, kinetic plots, temperature, speed 3 Correspondence:Professor Pat Sandra, Ghent University, Krijgslaan 281 S4-bis, B-9000, Ghent, Belgium E-mail: pat.sandra@richrom.comTel: +32 56 204031; Fax: +32 56 204858 IntroductionReversed-phase liquid chromatography (RP-LC) is by far the most widely employed analytical tool in drug discovery and development. In recent years, the efficiency of RP-LC has been drastically increased through various approaches including using sub-2µm particles in combination with ultra high pressure LC (UHPLC) [1][2][3], increasing the column length and operation at elevated temperature (ETLC) [4][5][6] or by combining both UHPLC and ETLC [7,8].These developments have certainly facilitated the development of generic methodologies for RP-LC for separations of active pharmaceutical ingredients (API's) and impurities [5].Furthermore, these advances have helped separation scientists to exploit early fundamental theories around the principles of separation optimisation with respect to efficiency and speed by utilisation of kinetic plots. For the pharmaceutical industry, which will certainly still have a plethora of conventional HPLC instrumentation in its good manufacturing practice (GMP) laboratories, kinetic plots can certainly serve a purpose in directing how conventional instrumentation and columns can be used more appropriately in combination with ETLC and long columns. This can help to optimize the use of conventional particle diameters (5 µm) to yield either maximum efficiency and to compared to sub-2µm particles, or to increased the speed of analysis.Since these developments, a chromatographic technique that has been enjoying recent attention in the pharmaceutical industry is hydrophilic interaction chromatography (HILIC).HILIC has become a valuable addition t...

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Combination of COFRADIC and high temperature – extended column length conventional liquid chromatography: A very efficient way to tackle complex protein samples, such as serum

Cited by 16 publications

References 30 publications

Tryptic digest analysis by comprehensive reversed phase×two reversed phase liquid chromatography (RP‐LC×2RP‐LC) at different pH's

Tryptic digest analysis by comprehensive reversed phase×two reversed phase liquid chromatography (RP‐LC×2RP‐LC) at different pH's

A strategy for the prior processing of high‐resolution mass spectral data obtained from high‐dimensional combined fractional diagonal chromatography

High-efficiency hydrophilic interaction chromatography by coupling 25cm×4.6mm ID×5μm silica columns and operation at 80°C

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