Reversed-phase liquid chromatography protein separation

Mao, Y., Zhang, X.M. (2003). Comprehensive two-dimensional separation system hy coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection. Electrophoresis 24, 3289-3295. 

As a rule, a separation method should be used for both purification and concentration of the sample. The classic method for peptides and proteins is a reverse-phase liquid chromatography preparation of the sample, followed by a concentration step (often lyophiliza-tion) of the fraction of interest. During those steps performed on very small quantities of sample, loss on the sample can occur if care is not taken to avoid it. Lyophilization, for instance, can lead to the loss of the sample absorbed on the walls of the vial. The use of separation methods on-line with the mass spectrometer often are preferred. Micro- or nano-HPLC [32,33] and capillary electrophoresis [34], both coupled mainly to electrospray ionization/mass spectrometry (ESI-MS), are used more and more. 

The ion-exchange constant does vary from one pair of ions to another therefore selectivity can be controlled by appropriate choice of the counter ion. However, selectivity is more conveniently manipulated by controlling the pH of the mobile phase and taking advantage of differences in the values of the analytes to be separated. In contrast with reversed-phase liquid chromatography (see section 3.6.2.1) the retention of weak acids and weak bases will reach a maximum when the compounds are in their ionized forms. Zwitterionic compounds such as amino acids, peptides and proteins can be separated on anion exchangers or cation exchangers. 

The first step is the priming of the NRPS active site and a subsequent limited tryptic digest of the protein. The digested sample is loaded on a reverse-phase liquid chromatography (RPLC) C18 column, which is directly connected to the inlet of an FT mass spectrometer. During online LC separation, the eluent is analyzed by MS and MS2 on an LC timescale. In the mass spectrometer the eluent is first analyzed by broadband Fourier transform mass spectrometry (FTMS). Then, peaks in the resulting broadband FT mass spectrum are... 

Comprehensive multidimensional liquid chromatography is a relatively new development and has yet to develop a diverse application base. For the time being applications are dominated by the separation of proteins and synthetic polymers. For proteins the first dimension separations are usually based on ion exchange and the second dimension separations on reversed-phase liquid chromatography. Gradient elution was often used for both separation modes with a separation time less than 2 minutes for the second dimension separation and from 30 minutes to several hours for the first dimension separation. Current trends include the use of non-porous particles and perfusive stationary phases for the second dimension separation to reduce the total separation time and wider internal diameter columns in place of packed capillary columns to simplify interface construction and instrument operation and to allow the loading of larger sample sizes. 

Reversed Phase-Liquid Chromatography (RP-HPLC) Separation of Proteins... 

Reversed-Phase Liquid Chromatography (RPLC) is an important tool in protein chemistry. Examination of sorption isotherms revealed that alcohohc buffers did not desorb proteins near physiological pH in RPLC systems, while buffers containing a poly(ethoxy alcohol) surfactant did not desorb protein at pH 2, but they did at pH 7 with concentrations of surfactant apparently well above the critical micellar concentration (cmc) [2]. It has been proposed that a necessary condition for the desorption of a protein from a surface is that the surface tension of the solvent falls between that of the protein and the surface [6]. This condition is fiilfilled for many proteins with surfactant solutions near conditions of physiological pH and ionic strength. Therefore, it was expected that separations of proteins could be thieved in these conditions. 

During the course of protein chemistry research looking for fluorescent compounds sensitive to the surrounding hydrophobicity, Weber designed l-dimethylaminonaphthalene-5-sulfonyl chloride (DNS-Cl) in 1952 [23]. DNS-Cl reacts with primary and secondary amino compounds (Figure 6.2) to form a fluorescent derivative with a yellow-green fluorescence [24]. DNS—amino acids can be separated by both normal-phase and reversed-phase liquid chromatography [25] and... 

Peptide mapping is a powerful tool for the analysis of the primary structure of a protein. This method typically takes advantage of one or more specific proteases that cleave the protein into smaller peptides, which are then separated and analyzed by reversed-phase liquid chromatography (RP-HPLC), sometimes in conjunction with mass spectrometry (MS). 

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