Influence of acid-induced conformational variability on protein separation in reversed phase high performance liquid chromatography

Bobály, Balázs; Tóth, Eszter; Drahos, László; Zsila, Ferenc; Visy, Júlia; Fekete, Jenő; Vékey, Károly

doi:10.1016/j.chroma.2013.12.022

Cited by 19 publications

(6 citation statements)

References 46 publications

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“…peaks increased in intensity with longer heating times. Bobály et al (2014), on the other hand, observed increased molar ellipticity values in CD spectra for lysozymes in acidic conditions, similar to what we observed in our systems at pH 3.5 and 6.5, heated to 75°C for 40 min (Fig. 3b).…”

Section: Circular Dichroismsupporting

confidence: 91%

Production, characterization and foamability of α-lactalbumin/glycomacropeptide supramolecular structures

Diniz¹,

Coimbra²,

Teixeira³

et al. 2014

Food Research International

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The study of protein interactions has generated great interest in the food industry. Therefore, research on new supramolecular structures shows promise. Supramolecular structures of the whey proteins α-lactalbumin and glycomacropeptide were produced under varying heat treatments (25 to 75°C) and acidic conditions (pH3.5 to 6.5). Isothermal titration calorimetry experiments showed protein interactions and demonstrated that this is an enthalpically driven process. Supramolecular protein structures in aqueous solutions were characterized by circular dichroism and intrinsic fluorescence spectroscopy. Additional photon correlation spectroscopy experiments showed that the size distribution of the structures ranged from 4 to 3545nm among the different conditions. At higher temperatures, lower pH increased particle size. The foamability of the supramolecular protein structures was evaluated. Analysis of variance and analysis of regression for foaming properties indicated that the two-factor interactions between pH and temperature exhibited a significant effect on the volume and stability of the foam.

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Section: Circular Dichroismsupporting

confidence: 91%

Production, characterization and foamability of α-lactalbumin/glycomacropeptide supramolecular structures

Diniz¹,

Coimbra²,

Teixeira³

et al. 2014

Food Research International

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“…As shown in Fig. 6, a baseline separation of four proteins was achieved within only seven min using a 2 min gradient of ACN from 3.2 to 30% in mobile phase, which is superior to the separation performance of RP monolithic column poly (AOD-co-EDMA) for same protein standards [29] and some packed RP columns [39]. It was also noticed that the peak shapes of all four proteins are sharp, indicating that there is little nonspecific protein adsorption when using this novel IAM monolithic column.…”

Section: Application Of the Poly(mdpc-co-edma) Monolithmentioning

confidence: 92%

Preparation of a biomimetic polyphosphorylcholine monolithic column for immobilized artificial membrane chromatography

Zhao

Chen

Zhou

et al. 2015

Journal of Chromatography A

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“…When liquid chromatography is online hyphenated to mass spectrometry, the advantages of using TFA for peptide separation quickly become significant disadvantages since the stable ion pairs and the increased surface tension of solutions with TFA prevent efficient ionization using electrospray. , Much effort has been spent on keeping TFA usable for LC-MS of peptides and proteins because of its superior chromatographic properties, particularly in RPLC of proteins, where it dramatically limits undesirable secondary interactions and maintain the compact structure of the analytes. − While the superior chromatographic performance of TFA, or recently its less fluorinated alternative difluoroacetic acid, added to the mobile phase at least in some percentage, may still predominate the signal suppression in protein LC-MS analyses, ,− modern stationary phases with reduced silanol activity and positive surface charge enable efficient separation of peptides only with formic acid.…”

Section: Rplc Methods For Bottom-up Proteomic Analysesmentioning

confidence: 99%

Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial

et al. 2022

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The performance of the current bottom-up liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses has undoubtedly been fueled by spectacular progress in mass spectrometry. It is thus not surprising that the MS instrument attracts the most attention during LC-MS method development, whereas optimizing conditions for peptide separation using reversed-phase liquid chromatography (RPLC) remains somewhat in its shadow. Consequently, the wisdom of the fundaments of chromatography is slowly vanishing from some laboratories. However, the full potential of advanced MS instruments cannot be achieved without highly efficient RPLC. This is impossible to attain without understanding fundamental processes in the chromatographic system and the properties of peptides important for their chromatographic behavior. We wrote this tutorial intending to give practitioners an overview of critical aspects of peptide separation using RPLC to facilitate setting the LC parameters so that they can leverage the full capabilities of their MS instruments. After briefly introducing the gradient separation of peptides, we discuss their properties that affect the quality of LC-MS chromatograms the most. Next, we address the in-column and extra-column broadening. The last section is devoted to key parameters of LC-MS methods. We also extracted trends in practice from recent bottom-up proteomics studies and correlated them with the current knowledge on peptide RPLC separation.

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Influence of acid-induced conformational variability on protein separation in reversed phase high performance liquid chromatography

Abstract: 26Influence of acid concentration in the mobile phase on protein separation was studied in a 27 wide concentration range of using trifluoroacetic acid (TFA) and formic acid (FA). At low,

Cited by 19 publications

References 46 publications

Production, characterization and foamability of α-lactalbumin/glycomacropeptide supramolecular structures

Production, characterization and foamability of α-lactalbumin/glycomacropeptide supramolecular structures

Preparation of a biomimetic polyphosphorylcholine monolithic column for immobilized artificial membrane chromatography

Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial

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