Fast high performance liquid chromatography separations for proteomic applications using Fused-Core? silica particles (original) (raw)
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Wider Pore Superficially Porous Particles for Peptide Separations by HPLC
Journal of Chromatographic Science, 2010
Fused-core superficially porous particles have recently created considerable interest for high-performance liquid chromatography separations because of their unusual high column efficiency and much lower back pressure when compared to sub-2-µm particles. With superficially porous particles, larger solutes can move rapidly in and out of a thin porous shell, resulting in reduced band broadening at higher mobile phase velocities for greater separation speeds. The original silica fused-core particles were 2.7 µm in diameter with a 0.5-µm thick shell of 90 Å pores designed for the fast separation of small molecules with molecular weights of less than approximately 5000. This manuscript describes new fused-core particles with similar physical characteristics except with a porous shell of 160 Å pores designed specifically for rapidly separating peptides (and some small proteins) with molecular weights up to approximately 15,000 Daltons. Because of the larger pore size, restricted diffusion of these larger molecules is not seen since ready access to the entire porous shell is featured. Data are given to define sample loading qualities for columns of these new particles. Column stability studies indicate that these particles bonded with a sterically protected C 18 stationary phase can be used at low pH and higher temperatures with excellent results. The wider-pore particles of this study are shown to be particularly useful with a mass spectrometer detector for the rapid gradient separation of peptides using both volatile trifluoroacetic acid and formic acid containing mobile phases. Examples are provided for the separation of complex peptide mixtures to illustrate the capabilities for columns of these new wider-pore, fused-core particles. Experimental Fused-core particles have been designed and prepared by Advanced Materials Technology, Inc., (Wilmington, DE). These high-purity Type B silica particles have an overall diameter of 2.7 μm, with a solid core diameter of 1.7 μm and an outer porous shell thickness of 0.5 μm. The new wider-pore particles have an average pore diameter of 160 Å (BET), compared with 90 Å pores for the original Halo particles. The surface area of these new particles is~80 m 2 /g, with a pore volume of~0.30 mL/g and an overall particle density of 1.3 cc/g. Columns of the wider-pore particles with 0.21-0.46 cm i.d. have been prepared by typical
Polymers
Separation with high efficiency and good resolution is constantly in demand in the pharmaceutical industry. The fast and efficient separation of complex samples such as peptides and proteins is a challenging task. To achieve high efficiency with good resolution, chromatographers are moving towards small particles packed into narrow-bore columns. Silica monolith particles (sub-2 µm) were derivatized with chlorodimethyl octadecyl silane (C18) and packed into stainless steel columns (100 mm × 1.8 mm i.d) by a slurry-packing method. The developed columns were used for the separation of peptides and proteins. A separation efficiency (N) of 40,000 plates/column (400,000 plates/m) was achieved for the mixture of five peptides. Similarly, the fast separation of the peptides was carried out using a high flow rate, and the separation of the five peptides was achieved in one minute with high efficiency (N ≅ 240,000 plates/m). The limit of detection (DL) and the limit of quantification (QL) for...
High peak capacity separation of peptides through the serial connection of LC shell-packed columns
Journal of Separation Science, 2009
High peak capacity separation of peptides through the serial connection of LC shell-packed columns High peak capacity was obtained for the separation of a HSA tryptic digest through the serial connection of three LC columns packed with shell material (C18, 15 cm64.6 mm, 2.7 lm particle size). A major benefit of these particles, consisting of a 1.7 lm solid core and a 0.5 lm porous shell, is the small diffusion path, which reduces axial dispersion of solutes and minimizes peak broadening allowing for higher resolving power to be obtained. Increase in the temperature decreases the viscosity and the backpressure on the columns and in this way allowed to couple multiple columns. Single and multicolumn systems were first evaluated for the separation of a standard sample mixture in the isocratic mode; afterwards three columns could be coupled at 608C, generating more than 90 000 effective plates (200 000 plates/meter) for the separation of HSA in gradient mode on a conventional HPLC instrument. Peak capacity ratios were found in good accordance with the theoretical gain in resolution, i.e. values of 1.37 and 1.67 were measured, when doubling and tripling the length of stationary phases, respectively (the highest measured peak capacity on three coupled columns was 367).
Journal of Chromatography B, 2009
Multidimensional liquid-based separation techniques are described for maximizing the resolution of the enormous number of peptides generated upon tryptic digestion of proteomes, and hence, reduce the spatial and temporal complexity of the sample to a level that allows successful mass spectrometric analysis. This review complements the previous contribution on unidimensional high performance liquid chromatography (HPLC). Both chromatography and electrophoresis will be discussed albeit with reversed-phase HPLC (RPLC) as the final separation dimension prior to MS analysis.
Journal of Chromatography A, 2000
Multimodal copolymer-encapsulated particles for liquid chromatography were prepared by bonding 1-octadecene and unsaturated carboxylic acids on silica particles (5 mm diameter, 300 A pores) for liquid chromatography of proteins. These multimodal copolymer-encapsulated particles can provide both hydrophobic and hydrogen bonding interactions with polar compounds. The chromatographic performance of these multimodal copolymer-encapsulated particles for peptide and protein separations was evaluated under reversed-phase conditions. Compared with typical C-bonded silica, polymer-encapsulated 8 6 particles were more stable in acidic mobile phases and provided better recoveries, especially for large proteins (M .0.5?10). r Totally hydrophobic polymer-encapsulated particles were found to produce broad peaks for proteins, and significant improvements were observed by introducing hydrophilic groups (-COOH) onto the polymer-encapsulated surface to form a multimodal phase. For the reversed-phase liquid chromatography of peptides and proteins, improved selectivity and increased solute retention were found using the multimodal polymer-encapsulated particles. More peaks were resolved for the separation of complex peptide mixtures such as protein digests using the multimodal polymer-encapsulated particles as compared to totally hydrophobic polymer-encapsulated particles.
Journal of Separation Science, 2007
Selective extraction of peptides in acidic human plasma by porous silica nanoparticles for peptidome analysis with 2-D LC-MS/MS In this study, an improved method for human plasma peptidome analysis including selective porous silica nanoparticles (MCM-41) extraction and subsequent online 2-D nano-LC-MS/MS analysis was established. Enhanced enrichment efficiency for the MCM-41 extraction was obtained by adjusting the pH of the plasma sample to 2.5. A total of 1680 unique peptides were identified in the plasma sample obtained from one healthy donor, which is nearly twice the amount identified from the native state of the plasma sample. The hydrophobic property, molecular weight (MW), and pI distribution of the identified peptides at pH 2.5 and native state of the plasma sample were systematically investigated and compared. Furthermore, many unusual cleaved peptides from plasma proteins (e. g., HSA) were observed at pH 2.5, which clearly show a ladder pattern. The cleavage patterns for all of the identified peptides at pH 2.5 were summarized, and chymosin and cathepsin D were confirmed as the possible peptidases responsible for the change of cleavage pattern in peptide profiling.
Journal of Chromatography A, 2003
In this paper, a general procedure is described for the generation of peptide maps of proteins with monolithic silica-based columns. The peptide fragments were obtained by tryptic digestion of various cytochrome c species with purification of the tryptic fragments achieved by reversed-phase high-performance liquid chromatographic methods. Peak assignment of the various peptides was based on evaluation of the biophysical properties of the individual peptides and via mass spectrometric identification. The performance of several different monolithic sorbents prepared as columns of identical cross-sectional dimensions were investigated as part of these peptide mapping studies and the data evaluated by applying solvent strength theory. These studies revealed curvilinear dependencies in the corresponding relative resolution maps. These findings directly impact on the selection of specific sorbent types or column configurations for peptide separations with silica rod monoliths. Moreover, the influence of variations in the amino acid sequence of the cytochrome cs were evaluated with respect to their effect on intrinsic hydrophobicity, the number of experimental observed tryptic cleavage sites, detection limits of the derived fragments in relation to their molecular size, and the chromatographic selectivity and resolution of the various peptides obtained following enzymatic fragmentation of the parent protein. Finally, the scope of these approaches in method development was examined in terms of robustness and efficiency.
Cation-exchange chromatography of peptides on poly(2-sulfoethyl aspartamide)-silica
Journal of Chromatography A, 1988
A strong cation-exchange material, poly(Zsulfoethy1 aspartamide)-silica (PolySULFOETHYL Aspartamide) was developed for purification and analysis of peptides by high-performance liquid chromatography. All peptides examined were retained at pH 3, even when the amino terminus was the only basic group. Peptides were eluted in order of increasing number of basic residues with a salt gradient. Capacity was high, as was selectivity and column efficiency. This new column material displays modest mixed-mode effects, allowing the resolution of peptides having identical charges at a given pH. The selectivity can be manipulated by the addition of organic solvent to the mobile phases; this increases the retention of some peptides and decreases the retention of others. The retention in any given case may reflect a combination of steric factors and non-electrostatic interactions. Selectivity was complementary to that of reversed-phase chromatography (RPC) materials. Excellent purifications were obtained by sequential use of Poly-SULFOETHYL Aspartamide and RPC columns for purification of peptides from crude tissue extracts. The new cation exchanger is quite promising as a supplement to RPC for general peptide chromatography.