Separation of intact proteins on γ-ray-induced polymethacrylate monolithic columns: A highly permeable stationary phase with high peak capacity for capillary high-performance liquid chromatography with high-resolution mass spectrometry (original) (raw)
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Journal of Chromatography A, 2011
The separation of intact proteins, including protein isoforms arising from various amino-acid modifications, employing a poly(styrene-co-divinylbenzene) monolithic capillary column in high-performance liquid chromatography coupled on-line to a time-of-flight mass spectrometer (MS) is described. Using a 250 mm × 0.2 mm monolithic capillary column high-sensitivity separations yielding peak capacities of >600 were achieved with a 2 h linear gradient and formic acid added in the mobile phase as ion-pairing agent. The combination of high-resolution chromatography with high-accuracy MS allowed to distinguish protein isoforms that differ only in their oxidation and biotinylation state allowing the separation between structural isoforms. Finally, the potential to separate proteins isoforms due to glycosylation is discussed.
Journal of Chromatography A, 2006
Preparation of organic polymer monolithic columns in fused silica capillaries was aimed at fast gradient separation of proteins. For this purpose, polymerization in situ procedure was optimized, using ethylene dimetacrylate and butyl metacrylate monomers with azobisisobutyronitrile as initiator of the polymerization reaction in presence of non-aqueous porogen solvent mixtures composed of 1-propanol and 1,4-butanediol. The separation of proteins in totally monolithic capillary columns was compared with the chromatography on a new type of "hybrid interparticle monolithic" capillary columns, prepared by in situ polymerization in capillary packed with superficially porous spherical beds, 37-50 m. The "hybrid" columns showed excellent stability and improved hydrodynamic flow properties with respect to the "totally" monolithic capillary columns. The separation selectivity is similar in the two types of columns. The nature of the superficially porous layer (bare silica or bonded C18 ligands) affects the separation selectivity less significantly than the porosity (density) of the monolithic moiety in the interparticle space, controlled by the composition of the polymerization mixture. The retention behaviour of proteins on all prepared columns is consistent with the reversed-phase gradient elution theory.
Journal of Separation Science, 2009
Original Paper 1 mm ID poly(styrene-co-divinylbenzene) monolithic columns for high-peak capacity one-and two-dimensional liquid chromatographic separations of intact proteins The LC performance of a 1650 mm polymer monolithic column format was demonstrated with high-peak capacity one-(1D) and offline two dimensional (2D) LC separations of intact proteins. After optimizing the RP 1D-LC conditions, including column temperature, flow rate and gradient time, a peak capacity of 475 was achieved within a 2-h analysis. The suitability of the monolithic column was also demonstrated for fast 1 min protein separations yielding 1 s peak widths determined at half peak height. In addition, an offline 2D-LC method was developed using the micro-fraction collection capabilities of the autosampler allowing automatic fractionation of intact proteins after the weak-ion-exchange (WAX) separation, and re-injection of the fractions onto the second-dimension RP monolithic column. The best peak capacity-to-analysis time ratio was obtained when applying 10 min seconddimension RP gradients. At optimized conditions, the WAX/6/RPLC separation of intact Escherichia coli proteins was performed within 6 h yielding a maximum theoretical peak capacity of 4880.
ELECTROPHORESIS, 2003
High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 mm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed highthroughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.
Indonesian Journal of Chemistry, 2010
Capillary column with monolithic stationary phase was prepared from silanized fused-silica capillary of 200 µm I.D. by in situ free radical polymerization of divinylbenzene with glycidy methacrylate in the presence of decanol and tetrahydrofuran as porogens. The hydrodynamic and chromatographic properties of this monolith, such as backpressure at different flow-rate, pore size distribution, van Deemter plot and the effect of varying gradient-rate were investigated. Poly(glycidyl methacrylate-divinylbenzene) monolithic capillary has been used successfully for the reversed-phase chromatographic separation of proteins.
Journal of Chromatography A, 2017
We added some comments in the text relating to the new literature references #66 and 67. 26. The performance of the monoliths presented in impressive for the separation of small molecules, which is generally not achieved by other research groups. But the main question is: (1) why do your monoliths perform so well (is it better homogeneity or are there no surface diffusion/gel porosity effects and why not?) and (2) if the monoliths perform so much better for small molecules does this mean that the performance of large molecule separations is also better. Discussion on these aspects is very much appreciated! We have not addressed these points in the paper, especially the relationship, if any, between the observed efficiency for small and large (bio)molecules. At this point, any comment would be speculative and we are planning to deeply investigate the interesting subject in a near future. 27. General comment: improve figure quality (4, 5, 6, 9).-labels on x and y axis, sometimes the font/size, sometimes the content!.-check the units.-check if appropriate scale are used (van Veemter curves: x-axis 5-6 mm/s does not show data points, kinetic plots y axis should be adjusted (there is no data in 10-1 and 10-2 range and 10-4 to 10-5 range, so please do not show this range). Corrected 28. Grammar last 2 highlights should be improved. Corrected Reviewer #2: Summary This work encompasses synthesis of poly(lauryl methacrylate-co-1,6-hexanediol dimethacrylate) monolithic columns in capillary diameters (75 µm, 200 µm and 250 µm i.d.) with a view to developing suitable materials for reversed-phase LC separation of small molecules, and to reinforce the suitability of the approach by demonstrating biomolecule separations. The novelty of the work is in the pre-treatment of capillary to allow a "grafting-to" approach to be used for anchoring the -ray initiated monolithic co-polymer to the surface. While no direct comparison to columns prepared using conventional pre-treatment procedures has been made within this work, the material has been structurally characterized (IR, solid state NMR, cryogenic NMR & SEM), compared to a thermally-initiated equivalent (using AIBN), with a number of chromatographic experiments with small and large molecules also shown. Isocratic chromatographic comparison centers around evaluation of newly-synthesized materials against a column packed with 5 µm C18functionalized particles in identical dimensions to the monolithic columns. Some further examples of biomolecule separations are shown in the gradient mode with high-resolution MS (Orbitrap). The monolith prepared is primarily novel due to the pre-treatment process used. Previous publications from this group (refs [17] & [43]) contain extensive examples of peptide and protein separations including peak capacity calculations. Thus, the biomolecule work is of secondary value, except that the best material is shown to demonstrate very similar characteristics as the same stationary phase material synthesized with a conventional pre-treatment process from previous publications. However, the performance for small molecule separations is excellent and shows some behavior that is contrast to previously-published materials of similar origin. Specifically the retention-dependence of plate height seems to be almost non-existent, which is a major problem for most organic polymer monoliths used for analytical chromatography of small molecules. There are some details missing from technical aspects of the work that I believe need to be addressed before publication. This includes a full explanation of the correction of plate height data for the chromatographic comparison (including the strange behavior of the packed column in terms We changed the Highlights accordingly, emphasizing the performances for both small molecules and proteins Abstract Line 41: has "reproducibility" been assessed here? Unless a different user, location, or instrument was the focus of this part of the study, then it is "repeatability"-e.g. batch-to-batch, column-tocolumn, run-to-run, interday… etc. Please choose accordingly. Changed Line 42: again, "methacrylate-based" would be better here. Changed Line 64: "They are most commonly obtained by a single-step…" Changed Line 70: "…suitable for high efficiency separations of large biomolecules,…" Changed Line 73: "…preparation and, consequently, monolithic…" Lines 77-78: "…used to implement the sensitivity of large molecules…" does not make sense. Please revise. Changed Line 79: "The majority of recent developments have been…" Changed Line 84: "microwave-assisted polymerization process." Line 85: missing comma after "photo-induced" Changed Line 408: "…for high speed and high efficiency separations…" Changed Line 414: "inspection" Changed Lines 417-419: see later comments on retention-dependence. A useful reference for comparison is:
Journal of Chromatography A, 2006
In the present work, an orthogonal two-dimensional (2D) capillary liquid chromatography (LC) method for fractionation and separation of proteins using wide range pH gradient ion exchange chromatography (IEC) in the first dimension and reversed phase (RP) in the second dimension, is demonstrated. In the first dimension a strong anion exchange (SAX) column subjected to a wide range (10.5-3.5) descending pH gradient was employed, while in the second dimension, a large pore (4000Å) polystyrene-divinylbenzene (PS-DVB) RP analytical column was used for separation of the protein pH-fractions from the first dimension. The separation power of the off-line 2D method was demonstrated by fractionation and separation of human plasma proteins. Seventeen pH-fractions were manually collected and immediately separated in the second dimension using a column switching capillary RP-LC system. Totally, more than 200 protein peaks were observed in the RP chromatograms of the pHfractions. On-line 2D analysis was performed for fractionation and separation of ten standard proteins. Two pH-fractions (basic and acidic) from the first dimension were trapped on PS-DVB RP trap columns prior to back-flushed elution onto the analytical RP column for fast separation of the proteins with UV/MS detection.
Journal of The American Society for Mass Spectrometry, 2012
Polypropylene (PP) capillary-channeled polymer (C-CP) films have parallel, μm-sized channels that induce solution wicking via capillary action. Efficient mass transport from the solution phase to the channel surface leads to adsorption of hydrophobic protein solutes. The basic premise by which C-CP films can be used as media to manipulate analyte solutions (e.g., proteins in buffer), for the purpose of desalting or chromatographic separation prior to MALDI-MS analysis is presented here. Cytochrome c and myoglobin prepared in a Tris-HCl buffer, and ribonuclease A, lysozyme, and transferrin prepared in phosphate buffered saline (PBS), are used as the test solutions to demonstrate the desalting concept. Protein analysis is performed after deposition on a C-CP film with and without a water washing step, followed by spray deposition of a typical sinapinic acid matrix. Extracted MALDI mass spectra exhibit much improved signal-to-noise characteristics after water washing. A mixture of cytochrome c and myoglobin (2 μL of 2.5 μM each in Tris-HCl buffer) was applied, washed with water and spatially separated via simple capillary action (wicking) using a reversed-phase solvent composition of 0.1% trifluoroacetic acid (TFA) in 50:50 acetonitrile (ACN):H 2 O. Subsequent application of sinapinic acid followed by imaging of the film using MALDI-MS reveals that as the protein solution is wicked down the film, separation occurs.