Biphasic Column Research Articles

Versatile ene-thiol photoclick reaction for preparation of multimodal monolithic silica capillary columns

This paper presents a photografting process of monolithic silica capillary columns based on the ene-thiol click chemistry. This study is performed on a “generic” vinyl-functionalized silica monolith (Hmin 6±1μm). The photoclick reaction is investigated using different thiol monomers (octadecanethiol, cysteine and sodium mercaptoethanesulfonate) to prepare capillary columns dedicated to various chromatographic modes (reversed-phase, HILIC and strong cation exchange). Whatever the monomer used, the photografting reaction is achieved in less than 5min with a relatively high thiol monomer content. This allows preparing highly retentive and efficient monolithic columns while avoiding polymerization and/or column clogging. In addition to the aforementioned properties (duration, versatility, efficiency), this photo-triggered chemical reaction allows addressing several appropriate surface functionalizations inside a single monolithic column in order to prepare nanovolume multimodal capillary columns. A multimodal biphasic monolithic column with a 1cm length cation-exchange segment followed by a 9cm length reversed-phase segment (SCX-RP) is prepared through two successive photografting reactions using a UV-mask to localize the reactions. This multimodal biphasic column is investigated using a model sample for the selective fractionation and separation of cationic and neutral compounds and is applied to the on-line preconcentration and separation of β-blockers.

Development of online high-/low-pH reversed-phase–reversed-phase two-dimensional liquid chromatography for shotgun proteomics: A reversed-phase-strong cation exchange-reversed-phase approach

Previously, we described an online high-/low-pH RP–RP LC system exhibiting high-throughput, automatability, and performance comparable with that of SCX-RP. Herein, we report a variant of the RP–RP platform, RP-SCX-RP, featuring an additional SCX trap column between the two LC dimensions. The SCX column in combination with the second-dimension RP can be used as an SCX-RP biphasic column for trapping peptides in the eluent from the first RP column. We evaluated the performance of the new platform through proteomic analysis of Arabidopsis thaliana chloroplast samples and mouse embryonic mouse fibroblast STO cell lysate at low-microgram levels. In general, RP-SCX-RP enhanced protein identification by allowing the detection of a larger number of hydrophilic peptides. Furthermore, the platform was useful for the quantitative analyses of crude chloroplast samples for iTRAQ applications at low-microgram levels. In addition, it allowed the online removal of sodium dodecyl sulfate and other chemicals used in excess in iTRAQ reactions, avoiding the need for time-consuming offline SCX clean-up prior to RP–RP separation. Relative to the RP–RP system, our newly developed RP-SCX-RP platform allowed the detection of a larger number of differentially expressed proteins in a crude iTRAQ-labeled chloroplast protein sample.

Automated Phosphoproteome Analysis for Cultured Cancer Cells by Two-Dimensional NanoLC-MS Using a Calcined Titania/C18 Biphasic Column

We have developed an on-line automated system for phosphoproteome analysis using titania-based phosphopeptide enrichment followed by nanoLC-MS/MS. Titania beads were prepared by calcination of commercial chromatographic titania beads at 800 degrees C to convert the crystalline structure. The obtained rutile-form titania exhibited higher selectivity in phosphopeptide enrichment than commercial titania, even in the absence of a competitive chelating reagent for non-phosphopeptides. For phosphoproteome analysis of human cervical cancer HeLa cells, tryptic digests of the cell extracts were directly injected into this on-line system, and 696 non-redundant phosphopeptides with 671 unambiguously determined phosphorylation sites, derived from 512 phosphoproteins, were successfully identified. This is the first successful application of an on-line automated phosphoproteome analysis system to complex biological samples.

Online Multidimensional Separation with Biphasic Monolithic Capillary Column for Shotgun Proteome Analysis

A biphasic monolithic capillary column with 10 cm segment of strong-cation exchange monolith and 65 cm segment of reversed-phase monolith was prepared within a single 100 microm i.d. capillary. Separation performance of this column was evaluated by a five-cycle online multidimensional separation of 10 microg tryptic digest of yeast proteins using nanoflow liquid chromatography coupled with tandem mass spectrometry, and it took 12 h for whole separation under the operating pressure only approximately 900 psi. Totally, 780 distinct proteins were positively identified through assignment of 2953 unique peptides at false-positive rate less than 1%. The good separation performance of this biphasic column was largely attributed to the good orthogonality of the strong-cation exchange monolith and reversed-phase monolith for multidimensional separation.

Multidimensional Protein Identification Technology (MudPIT) Analysis of Ubiquitinated Proteins in Plants

Protein conjugation with ubiquitin, known as ubiquitination, is a key regulatory mechanism to control protein abundance, localization, and activity in eukaryotic cells. To identify ubiquitin-dependent regulatory steps in plants, we developed a robust affinity purification/identification system for ubiquitinated proteins. Using GST-tagged ubiquitin binding domains, we performed a large scale affinity purification of ubiquitinated proteins from Arabidopsis cell suspension culture. High molecular weight ubiquitinated proteins were separated by SDS-PAGE, and the trypsin-digested samples were then analyzed by a multidimensional protein identification technology (MudPIT) system. A total of 294 proteins specifically bound by the GST-tagged ubiquitin binding domains were identified. From these we determined 85 ubiquitinated lysine residues in 56 proteins, confirming the enrichment of the target class of proteins. Our data provide the first view of the ubiquitinated proteome in plants. We also provide evidence that this technique can be broadly applied to the study of protein ubiquitination in diverse plant species.

HPLC Separation of Digested Proteins and Preparation for Matrix-Assisted Laser Desorption/Ionization Analysis

INTRODUCTIONTwo types of columns are commonly used for the separation of peptides by HPLC. A single-phase column contains the reverse-phase resin C18, which interacts with the hydrophobic moieties of the peptides. Peptides resulting from digestion of simple mixtures of proteins are loaded onto the single-phase column and eluted into the mass analyzer using an increasing gradient of an organic solvent. Peptides resulting from the digestion of more complex mixtures of proteins are resolved using a biphasic column. This column integrates both a strong cation exchange SCX resin, which interacts with peptides as a result of their positive charge, and a reverse-phase C18 resin, packed in tandem. Peptides initially interact with the SCX resin and are eluted into the C18 resin by ammonium acetate that competes for the peptide-binding sites. Peptides are then eluted from the C18 resin into the mass analyzer. This process is repeated using increasing concentrations of ammonium acetate to differentially elute peptides in a stepwise fashion. The biphasic column also uses an additional C18 reverse-phase resin linked through an Inline MicroFilter Assembly (Upchurch) to desalt the peptides prior to loading onto the SCX. The desalting and biphasic columns are combined to give an integrated desalting/biphasic column.

Direct Enzymatic Digestion of Protein Complexes for MS Analysis

INTRODUCTIONThe individual components of a purified protein complex can be determined by MS. Components can be separated by SDS-PAGE and subsequently digested, or the entire protein complex can be digested by chemical agents or by proteases. The mixture of peptides that results from digestion without prior separation is more complex than that obtained from the digestion of a single protein, and can best be resolved subsequently using HPLC in combination with a biphasic column. Protein complexes can be digested directly using several different protocols. Common methods include a double digest with endoproteinase Lys-C and trypsin, or a triple digest with elastase, subtilisin, and trypsin. Endoproteinase Lys-C cleaves carboxy-terminal to lysine residues, while trypsin cleaves carboxy-terminal to lysine and arginine residues. The triple digest uses the relatively nonspecific proteases elastase and subtilisin to create a large number of overlapping peptides for the analysis of post-translational modifications. The double and triple digest protocols begin with the reduction and alkylation of cysteine residues to disrupt disulfide bonds and, therefore, higher-order protein structure.

Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: single-dimension LC-MS/MS, 2-phase MudPIT, and 3-phase MudPIT

One of the most effective methods for the direct identification of proteins from complex mixtures without first having to resolve them by polyacrylamide gel electrophoresis is to separate proteolytically generated peptides by microcapillary HPLC and then collect data directly on the eluent using a tandem mass spectrometer. Multidimensional HPLC separation techniques provide access to even more complex mixtures of proteins. A set of techniques for multidimensional analysis was developed in our lab; collectively they are known as multidimensional protein identification technology (MudPIT). These strategies employ a biphasic column with a section of reversed phase (RP) material flanked by strong cation exchange (SCX) resin and allow for multidimensional separation of peptides. A variation on MudPIT adds an additional section of RP material behind the SCX and RP. This 3-phase column can be used for “online” desalting of the sample. We compare the analysis of a complex mixture of proteins purified by their association with bovine brain microtubules using a single-dimension LC-MS/MS column, a 2-phase (standard) MudPIT column, and a 3-phase MudPIT column. We find that the 3-phase MudPIT column yields a greater number of protein identifications for this test sample and allows data to be collected on a set of hydrophilic peptides not sampled using the 2-phase MudPIT column.

An automated multidimensional protein identification technology for shotgun proteomics.

We describe an automated method for shotgun proteomics named multidimensional protein identification technology (MudPIT), which combines multidimensional liquid chromatography with electrospray ionization tandem mass spectrometry. The multidimensional liquid chromatography method integrates a strong cation-exchange (SCX) resin and reversed-phase resin in a biphasic column. We detail the improvements over a system described by Link et al. (Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676-682) that separates and acquires tandem mass spectra for thousands of peptides. Peptides elute off the SCX phase by increasing pI, and elution off the SCX material is evenly distributed across an analysis. In addition, we describe the chromatographic benchmarks of MudPIT. MudPIT was reproducible within 0.5% between two analyses. Furthermore, a dynamic range of 10000 to 1 between the most abundant and least abundant proteins/peptides in a complex peptide mixture has been demonstrated. By improving sample preparation along with separations, the method improves the overall analysis of proteomes by identifying proteins of all functional and physical classes.

Adsorptive Biphasic Column Technology for Protein Sequence Analysis and Protein Chemical Modification

The Hewlett-Packard adsorptive, biphasic column technology serves as the basis for the development of exceedingly efficient automated N-terminal protein sequence analysis and automated protein chemical modifications. The technology enables the facile capture of protein and peptide samples recovered from various isolation and purification protocols directly onto miniature hydrophobic, reversed-phase sample columns that are then subjected to N-terminal sequence analysis using the HP G1005A Protein Sequencing System or protein chemical modifications using the HP G1004B Protein Chemistry Station. The process minimizes the sample losses and manipulations routinely associated with alternative analytical procedures. Protein and peptide samples, prepared in amine-containing buffers, detergent systems, chaotropic solutions, inorganic salt solutions, organic solvents, and any other typically encountered sample matrix, are amenable for direct application on the reversed-phase sample column, which adsorptively immobilizes the samples but permits the elimination of interfering buffer and matrix components prior to sequence analysis or chemical modification. The N-terminal sequencing strategy invokes the use of a hydrophilic, strong-anion-exchange column mated to the reversed-phase sample column to assemble a complete protein sequencer column. The biphasic properties of the protein sequencer column result in an optimization of the Edman-based protein sequencing chemistry. The integration of automated column-based protein chemical modification with automated column-based protein sequence analysis provides a powerful methodology for obtaining extensive protein structural information without the risks and complications of intervening tedious and costly sample handling steps.

Simultaneous determination of adreno-ovarian steroids from a single aliquot

A technique standardised for simultaneous fractionation and estimation of major adreno-ovarian steroids has been applied to biological samples in midpregnancy in guinea pigs. The hormones have been separated on a single TLC plate using single solvent system and measured on a single biphasic column on the basis of a single aliquot. The technique is considerably helpful for investigations of complexities of the adreno-genital syndrome. Ovarian and blood plasma progesterone showed significant increase of 76–77% in pregnancy. Estrone level of plasma in pregnancy indicated significant increase of 34%. Estradiol and estriol in blood plasma in non-pregnancy, and estriol in placenta were not detectable. Urinary estradiol was significantly increased in pregnancy. Cortisol level in adrenals, and that of cortisone and cortisol in blood plasma were significantly higher during pregnancy. Cortisol and estriol were not adequately separated by TLC, however, subsequent estimation by GLC obviated the overlapping effects because of different operating conditions of the two hormones.