Mixed‐mode hydrophilic interaction/cation‐exchange chromatography: Separation of complex mixtures of peptides of varying charge and hydrophobicity

Abstract

Mixed-mode hydrophilic interaction/cationexchange chromatography: Separation of complex mixtures of peptides of varying charge and hydrophobicity Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) was applied to the separation of two mixtures of synthetic peptide standards: (i) a 27-peptide mixture containing three groups of peptides (each group containing nine peptides of the same net charge of +1, +2 or +3), where the hydrophilicity/ hydrophobicity of adjacent peptides within the groups varied only subtly (generally by only a single carbon atom); and (ii) peptide pairs with the same composition but different sequences, where the sole difference between the peptides was the position of a single amino acid substitution. HILIC/CEX is essentially CEX chromatography in the presence of high levels of organic modifier (generally ACN). The present study demonstrated the dramatic effect of increasing ACN concentration (optimum levels of 60 -80%, depending on the application) on the separation of both mixtures of peptides. The greater the charge on the peptides, the better the separation achievable by HILIC/CEX. In addition, HILIC/CEX separation of both the peptide mixtures used in the present study was shown to be superior to that of the more commonly applied RP-HPLC mode. Our results highlight again the efficacy of HILIC/CEX as a peptide separation mode in its own right as well as an excellent complement to RP-HPLC. IntroductionMixed mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) is an HPLC approach to peptide separations introduced by our laboratory over 15 years ago [1]. HILIC/CEX combined the most advantageous aspects of two widely different separation mechanisms: a separation based on hydrophilicity/hydrophobicity differences between peptides overlaid on a separation based on net charge [1 -8]. Characteristic of HILIC/ CEX separations is the presence of a high organic modifier concentration (generally, ACN) to promote hydrophilic interactions between the solute and the hydrophilic/charged CEX stationary phase, with peptides then eluted from the column with a salt gradient. Peptides are generally eluted in groups of peptides in order of increasing net positive charge; within these groups, peptides are eluted in order of increasing hydrophilicity (decreasing hydrophobicity). Indeed, HILIC/CEX is basically CEX in the presence of high concentrations of ACN (60 -80%, in general).The HILIC/CEX approach has proven to be very versatile for applications to peptide separations, representing an excellent complement to RP-HPLC and, indeed rivalling or even exceeding RP-HPLC for specific peptide mixtures [2,3,6,9]. For example, HILIC/CEX has successfully separated deletion products from crude synthetic peptides (not achievable by RP-HPLC) [3] as well as enabled the purification of synthetic peptides from closely related serine side-chain acetylated peptides (again not achievable by RP-HPLC) [6]. The complementary nature of HILIC/ CEX and RP-HPLC was also demonstrated by the ...

Elution Profile of Di-peptides on a Sulfonated Ethylstyrene-Divinylbenzene Copolymer Resin Column by High-performance Liquid Chromatography

“…[1][2][3][4][5][6]16 The use of a cation-exchange/reversed phase column was attempted in the separation of acetylated oligo-(>10 mer) peptides, and a sufficient separation was achieved by hydrophilic interaction (HILIC) chromatography with increased hydrophilicity and salt (NaClO4) concentrations. 20 However, these reports were limited to the separation of terminal-protected or hydrophobic oligo-peptides, and did not include the separation of non-protected small (di-/tri-) peptides with varying pI profiles. The cation-exchange/reversed phase HPLC column used in this study was based on ethylstyrene-divinylbenzene copolymer resin, which permitted a dynamic pH-gradient elution of the mobile phase (in this study, pH 4.8 -8.9) for peptides with varying pI values.…”

Influence of stationary phase chemistry and mobile‐phase composition on retention, selectivity, and MS response in hydrophilic interaction chromatography

“…In addition, since the injection solvent used in HILIC is the same as the final solvent of many sample preparation techniques (liquid-liquid extraction, protein precipitation, and solid phase extraction), the need for evaporation and reconstitution of the extract is eliminated, thus increasing throughput of the overall assay. The interest in and practice of HILIC is anticipated to grow even more due to the increasing need to analyze highly polar molecules in all areas of science, including bioanalysis [3], proteomics [4,5], biopharmaceuticals, toxicology, drug metabolism and metabolic profiling, and other areas in the food and pharmaceutical industries [6][7][8][9]. HILIC has also been used as an orthogonal separation step in multidimensional chromatography [10,11].…”

Hydrophilic properties as a new contribution for computer-aided identification of short peptides in complex mixtures

“…The growing interest in HILIC is partly due to its high solvent compatibility with MS in the electrospray ionization (ESI) mode [35,36]. One of the major fields currently under investigation is the use of HILIC as a separation tool for proteomic applications [33,[36][37][38], but it is also studied for use in mixed-mode [39][40][41] and two-dimensional liquid chromatography (2D LC) [42][43][44][45].…”