Protein-induced conformational change in glycans decreases the resolution of glycoproteins in hydrophilic interaction liquid chromatography.

An understanding of why hydrophilic interaction liquid chromatography gives a higher resolution for glycans than for glycoproteins would facilitate column improvements. Separations of the glycoforms of ribonuclease B compared to its released glycans were studied using a commercial hydrophilic interaction liquid chromatography column. The findings were used to devise a new hydrophilic interaction liquid chromatography column. For the commercial column, chromatograms and van Deemter plots showed that selectivity and efficiency are comparable factors in the higher resolution of the released glycans. The higher selectivity for the released glycans was associated with more water molecules displaced per added mannose. To investigate why, three-dimensional structures of the glycoprotein and the glycan were computed under chromatographic conditions. These showed that hydrogen bonding within the free glycan makes its topology more planar, which would increase contact with the bonded phase. The protein sterically blocks the hydrogen bonding. The more globular-shaped glycan of the glycoprotein suggests that a thicker bonded phase might improve selectivity. This was tested by making a column with a copolymer bonded phase. The results confirmed that selectivity is increased. The findings are possibly broadly relevant to glycoprotein analysis since the structural motif involved in internal hydrogen bonding is common to N-linked glycans of human glycoproteins.

Evaluation of particle and bed integrity of aqueous size-exclusion columns packed with sub-2 µm particles operated at high pressure.

The performance of columns packed with 1.7 µm particles for aqueous size-exclusion chromatography was assessed at high-pressure conditions and linked to particle- and column-bed integrity. Decreasing the particle size from 3.5 µm to 1.7 µm increases the resolution due to the improved mass-transfer characteristics, allowing to significantly speed-up analysis without compromising the selectivity. A sub-minute separation of intact proteins was realized on a 4.6 mm i.d × 75 mm long column packed with 1.7 µm SEC particles applying a flow rate of 1.8 mL/min, corresponding to a column pressure of 530 bar. Ultra-high pressure operation (exceeding manufacturer's recommendations) resulted in peak deformation, a shift towards earlier retention times, and an alteration in selectivity. To gain insights in the mechanisms of column deterioration, short 30 mm long columns were operated at UHPLC conditions, maximizing the pressure drop over individual particles. This resulted in the presence of fractured particles situated at the column outlet, as verified by scanning electron micrographs. Mercury-intrusion porosimetry and argon-adsorption measurements did not reveal significant differences in intraparticle volume between particle batches sampled before and after pressure stress testing. As particles at the column outlet fracture (but not collapse) at high pressure operation, a void was formed at the column inlet. The degradation of the separation performance appeared to be the result of a decrease in interparticle pore volume.

Fluorescent Probes for Monitoring Serine Ubiquitination.

In a radical departure from the classical E1-E2-E3 three-enzyme mediated ubiquitination of eukaryotes, the recently described bacterial enzymes of the SidE family of Legionella pneumophila effectors utilize NAD+ to ligate ubiquitin onto target substrate proteins. This outcome is achieved via a two-step mechanism involving (1) ADP ribosylation of ubiquitin followed by (2) phosphotransfer to a target serine residue. Here, using fluorescent NAD+ analogues as well as synthetic substrate mimics, we have developed continuous assays enabling real-time monitoring of both steps of this mechanism. These assays are amenable to biochemical studies and high-throughput screening of inhibitors of these effectors, and the discovery and characterization of putative enzymes similar to members of the SidE family in other organisms. We also show their utility in studying enzymes that can reverse and inhibit this post-translational modification.

Making Sharper Peaks for Reverse-Phase Liquid Chromatography of Proteins.

Protein separations have gained increasing interest over the past two decades owing to the dramatic growth of proteins as therapeutics and the completion of the Human Genome Project. About every decade, the field of protein high-performance liquid chromatography (HPLC) seems to mature, having reached what appears to be a theoretical limit. But then scientists well versed in the basic principles of HPLC invented a way around the limit, generating another decade of exciting progress. There is still the need for higher resolution and better compatibility with mass spectrometry because it is an essential tool for identification of proteins and their modifications. To make advances, the fundamental principles need to be understood. This review covers recent advances and current needs in the context of the principles that underlie the many contributions to peak broadening.

Native Reversed-Phase Liquid Chromatography: A Technique for LCMS of Intact Antibody-Drug Conjugates.

The synthesis of antibody-drug conjugates (ADCs) using the interchain cysteines of the antibody inherently gives a mixture of proteins with varying drug-to-antibody ratio. The drug distribution profiles of ADCs are routinely characterized by hydrophobic interaction chromatography (HIC). Because HIC is not in-line compatible with mass spectrometry (MS) due to the high salt levels, it is laborious to identify the constituents of HIC peaks. An MS-compatible alternative to HIC is reported here: native reversed phase liquid chromatography (nRPLC). This novel technique employs a mobile phase 50 mM ammonium acetate for high sensitivity in MS and elution with a gradient of water/isopropanol. The key to the enhancement is a bonded phase giving weaker drug-surface interactions compared to the noncovalent interactions holding the antibody-drug conjugates together. The hydrophobicity of the bonded phase is varied, and the least hydrophobic bonded phase in the series, poly(methyl methacrylate), is found to resolve the intact constituents of a model ADC (Ab095-PZ) and a commercial ADC (brentuximab vedotin) under the MS-compatible conditions. The nRPLC-MS data show that all species, ranging from drug-to-antibody ratios of 1 to 8, remained intact in the column. Another desired advantage of the nRPLC is the ability of resolving multiple positional isomers of ADC that are not well-resolved in other chromatographic modes. This supports the premise that lower hydrophobicity of the bonded phase is the key to enabling online nRPLC-MS analysis of antibody-drug conjugates.

Electrophoresis of megaDalton proteins inside colloidal silica.

With the growth of the biopharmaceutical industry, there is a need for rapid size-analysis of proteins on the megaDalton scale. The large pore sizes needed for such separations cannot be easily reached by pushing the current limits of size-exclusion chromatography or gel electrophoresis. The concept detailed here is the formation of arbitrarily wide pores by packing nonporous colloidal silica in capillaries. This method can be called packed-capillary electrophoresis, or "pCE". Electrophoresis of protein standards (11-155 kDa) by pCE, using 345 nm diameter particles in 100 µm diameter capillaries, gives 2x higher resolution than a typical PAGE gel in 1/6 of the time. The electropherograms show that pCE is highly efficient, with half-micrometer plate heights for all seven standards, giving 105 plates for a 50 mm length. The large pore radius of 65 nm enables baseline resolution of proteins of 0.72, 1.048 and 1.236 MDa in less than 15 min. The short separation time of pCE is attributed to the absence of small pores that restrict protein migration in gels. The pCE separation is applied to the analysis of a stressed pharmaceutical-grade IgG4 sample, giving unprecedented baseline resolution of monomer, dimer, trimer and tetramer in less than 10 min.

Ubiquitin Chains Modified by the Bacterial Ligase SdeA Are Protected from Deubiquitinase Hydrolysis.

The SidE family of Legionella pneumophila effectors is a unique group of ubiquitin-modifying enzymes. Along with catalyzing NAD+-dependent ubiquitination of certain host proteins independent of the canonical E1/E2/E3 pathway, they have also been shown to produce phosphoribosylated free ubiquitin. This modified ubiquitin product is incompatible with conventional E1/E2/E3 ubiquitination processes, with the potential to lock down various cellular functions that are dependent on ubiquitin signaling. Here, we show that in addition to free ubiquitin, Lys63-, Lys48-, Lys11-, and Met1-linked diubiquitin chains are also modified by SdeA in a similar fashion. Both the proximal and distal ubiquitin moieties are targeted in the phosphoribosylation reaction. Furthermore, this renders the ubiquitin chains unable to be processed by a variety of deubiquitinating enzymes. These observations broaden the scope of SdeA's modulatory functions during Legionella infection.

Effect of immobilization on the antimicrobial activity of a cysteine-terminated antimicrobial Peptide Cecropin P1 tethered to silica nanoparticle against E. coli O157:H7 EDL933.

Antimicrobial peptides (AMPs) have the ability to penetrate the cell membrane, form pores which eventually lead to cell death. Immobilization of AMP on nanoparticles can play a major role in antimicrobial materials, biosensors for pathogen detection and in food safety. The minimum inhibitory concentration (MIC) of free Cecropin P1 (CP1, sequence SWLSTAKKLENSAKKRLSEGIAIAIQGGPR) and adsorbed on silica nanoparticle against E. coli O157:H7 EDL933 were 0.78µg/ml. This was found to be consistent with preservation of α-helical secondary structure of CP1 upon adsorption as indicated by circular dichroism (CD). Cysteine-terminus modified Cecropin P1 (CP1C, sequence SWLSTAKKLENSAKKRLSEGIAIAIQGGPRC) was chemically immobilized onto silica nanoparticles with maleimide-PEG-NHS ester cross-linkers of different PEG chain lengths. The antimicrobial activity of CP1C in solution and adsorbed on silica nanoparticles against E. coli O157:H7 EDL933 were found to be the same as those for CP1. However, tethered CP1C exhibited much higher MIC of 24.38, 37.55 and 109.82µg/ml for (PEG)20, (PEG)6 and (PEG)2 linkers respectively. The antimicrobial activity of CP1C tethered to silica nanoparticles with (PEG)20 linker was found to be lower for lower surface coverage with MIC values being 86.06, 36.89, 24.38 and 17.84µg/ml for surface coverage of 12.3%, 24.4%, 52.8% and 83.8% respectively. All atom MD simulation of 1:3 DOPG/DOPC mixed membrane interacting with free and PEGlyated CP1C indicated that presence of PEG linker prevented CP1C from interacting with the bilayer which may explain the loss of antimicrobial activity of tethered CP1C.

In-column bonded phase polymerization for improved packing uniformity.

It is difficult to pack chromatographic particles having polymeric-bonded phases because solvents used for making a stable slurry cause the polymer layer to swell. Growth of the polymer inside the column (in situ) after packing was investigated and compared with conventional, ex situ polymer growth. The method of activators generated by electron transfer, along with atom-transfer radical polymerization, enabled polymerization under ambient conditions. Nonporous, 0.62 µm silica particles with silane initiators were used. Polyacrylamide films with a hydrated thickness of 23 nm in 75:25 water/isopropanol grew in 55 min for both in situ and ex situ preparations, and the same carbon coverage was observed. Higher chromatographic resolution and better column-to-column reproducibility were observed for in situ polymer growth, as evaluated by hydrophilic interaction liquid chromatography for the model glycoprotein, ribonuclease B. In situ polymer growth was also found to give lower eddy diffusion, as shown by a narrower peak width for injected acetonitrile in 50:50 acetonitrile/water. When columns were packed more loosely, bed collapse occurred quickly for ex situ, but not for in situ, polymer growth. The higher resolution and stability for in situ polymer growth is explained by packing with hard, rather than soft, contacts between particles.

Chromatographic efficiency and selectivity in top-down proteomics of histones.

Histones are involved in epigenetic control of a wide variety of cellular processes through their multiple post-translational modifications. Their strongly cationic nature makes them challenging to separate with reversed-phase liquid chromatography coupled to mass spectrometry (RPLC-MS), where trifluoroacetic acid is avoided due to adduct formation. Columns with higher resolution are needed. In this work, RPLC-MS is performed on a histone sample using difluoroacetic acid and a 20-min gradient. Columns with C18 surfaces are compared for two different types of particle morphologies: 1) fully porous particles of 5µm in diameter, 2) superficially porous particles of 3µm in diameter with a shell of 0.2µm. The resolution for the histone separation is better for the latter column, but only when the modifier is trifluoroacetic acid, which is used with UV absorbance detection. When difluoroacetic acid is used for LCMS, the peaks broaden enough to erase the advantage in efficiency for the superficially porous particles. The fully porous and superficially porous cases show similar performance in RPLC-MS, with slightly higher resolution for the fully porous particles. The expected advantage of the shorter diffusion distances for the superficially porous particles is shown to be outweighed by the lower selectivity of its bonded phase.

Alleviating nonlinear behavior of disulfide isoforms in the reversed-phase liquid chromatography of IgG2.

Reversed-phase chromatography is an established method for characterizing the disulfide isoforms of IgG2. This work explores the effect of mobile phase gradient profile and sample concentration on the separation of disulfide isoforms. The acidic mobile phase can alter the relative proportions of disulfide isoforms, but only when the level of the reactive A1 isoform is much higher than for typical conditions of separation and typical IgG2 samples. Otherwise, there is minimal disulfide scrambling. A slower gradient and flow rate modestly improve resolution, but the peaks remain heavily overlapped. Resolution is further improved and nonlinear chromatography lessened when injection is performed under non-stacking conditions. Non-stacking conditions also keep the concentration from spiking at the head of the column, reducing noncovalent associations that can promote disulfide scrambling. The higher resolution from non-stacking injection reveals the presence of at least seven species.

Submicrometer particles and slip flow in liquid chromatography.

Smaller particles have progressively led to higher efficiency in liquid chromatography, particularly for proteins, due to smaller diffusion distances. Particle diameter has recently entered the submicrometer region, with the back-pressure requirements alleviated by slip flow.

Efficient separations of intact proteins using slip-flow with nano-liquid chromatography-mass spectrometry.

A capillary with a pulled tip, densely packed with silica particles of 0.47 µm in diameter, is shown to provide higher peak capacity and sensitivity in the separation of intact proteins by reversed-phase liquid chromatography-mass spectrometry (LC-MS). For a C18 bonded phase, slip flow gave a 10-fold flow enhancement to allow for stable nanospray with a 4-cm column length. Model proteins were studied: ribonuclease A, trypsin inhibitor, and carbonic anhydrase, where the latter had impurities of superoxide dismutase and ubiquitin. The proteins were well separated at room temperature with negligible peak tailing. The peak capacity for ubiquitin was 195 for a 10-min gradient and 315 for a 40-min gradient based on Gaussian fitting of the entire peak, rather than extrapolating the full-width at half-maximum. Separation of a cell lysate with a 60 min gradient showed extremely high peak capacities of 750 and above for a peptide and relatively homogeneous proteins. Clean, low noise mass spectra for each model protein were obtained. The physical widths of the peaks were an order of magnitude narrower than those of conventional columns, giving increased sensitivity. All proteins except ubiquitin exhibited significant heterogeneity apparently due to multiple proteoforms, as indicated by both peak shapes and mass spectra. The chromatograms exhibited excellent reproducibility in retention time, with relative standard deviations of 0.09 to 0.34%. The results indicate that submicrometer particles are promising for improving the separation dimension of LC in top-down proteomics.

RPLC of intact proteins using sub-0.5 µm particles and commercial instrumentation.

This paper addresses whether one can gain an improvement in speed or resolution with a silica colloidal crystal (SCC) of nonporous 470 nm particles when using a commercial nano-UHPLC. Compared to a capillary packed with nonporous 1.3 µm particles and the same C4 bonded phase, the peak width for BSA is decreased by a factor of 6.8 for the SCC. Some of this improvement is attributable to slip flow since the ratio of particle diameters is only 2.8. Resolution in protein separations was compared for a 2-cm capillary of SCC vs a 5-cm column of porous 1.7 µm particles. Both used a C4 bonded phase, and on-column fluorescence detection was used for the SCC. Split flow (5:1) before the SCC decreased the gradient delay time to 0.4 min and the injected volume to 0.4 nL. For variants from the labeling of BSA, the SCC had a 5-fold higher speed and 2-fold higher resolution than did the commercial column. For a monoclonal antibody and its aggregates, the SCC had a 3-fold higher speed and a 3-fold higher resolution compared to the commercial column. The SCC gave baseline resolution of the monomer, dimer and trimer in 5 min. The results show that a significant advantage can be gained using a commercial instrument with the SCC, despite the instrument not being designed for use with such small particles.

Polyacrylamide brush layer for hydrophilic interaction liquid chromatography of intact glycoproteins.

A chromatographic column of nonporous silica particles with a bonded phase of linear polyacrylamide chains is evaluated for hydrophilic interaction liquid chromatography (HILIC) of intact glycoproteins. The column is shown to retain glycoproteins significantly more strongly than non-glycoproteins. A particle diameter of 700nm gives two-fold higher resolution than does a 1.4µm particle diameter, and the column efficiency is found to be mostly limited by packing heterogeneity. LCMS is able to resolve the five glycoforms of ribonuclease B and give high quality mass spectra, but there is loss of resolution of the isomers of glycoforms due to the lower amount of TFA. Compared to two leading commercial HILIC columns operated at 60°C, the polyacrylamide column operated at 30°C provided at least two-fold higher resolution for intact ribonuclease B, and showed peaks for glycoforms of prostate specific antigen, although not resolved.

Silica colloidal crystals as emerging materials for high-throughput protein electrophoresis.

Silica colloidal crystals are a new type of media for protein electrophoresis, and they are assessed for their promise in rapidly measuring aggregation of monoclonal antibodies. The nature of silica colloidal crystals is described in the context of the need for a high-throughput separation tool for optimizing the formulations of protein drugs for minimal aggregation. The fundamental relations between molecular weight and mobility in electrophoresis are used to make a theoretical comparison of selectivity between gels and colloidal crystals. The results show that the selectivity is similar for these media, but slightly higher, 10%, for gels, and the velocity is inherently lower than that for gels due to the smaller free volume fraction. These factors are more than compensated for by lower broadening in colloidal crystals. These new media give plate heights of only 0.15 µm for the antibody monomer and 0.42 µm for the antibody dimer. The monoclonal antibody is separated from its dimer in 72 s over a distance of only 6.5 mm. This is five times faster than size-exclusion chromatography, with more than tenfold miniaturization, and amenable to parallel separations, all of which are promising for the design of high-throughput devices for optimizing protein drug formulations.

Insights from theory and experiments on slip flow in chromatography.

Slip flow has become a topic of interest in reversed-phase liquid chromatography because it gives a flow enhancement that facilitates the use of submicrometer particles, providing a large improvement in separation efficiency. Moreover, slip flow provides an additional improvement in efficiency by reducing the velocity distribution in the mobile phase. The phenomenon of slip flow in open tubes is described in chromatographically relative terms. A recent paper in this journal is discussed, as it provides the first theoretical study of slip flow in packed beds, in this case for face-centered cubic geometry. The theory paper reveals that the presence of the packed bed introduces a heterogeneity in fluid velocities that is absent in open tubes, reducing the additional improvement in efficiency from slip flow. The recent paper also suggests that there is yet another factor improving efficiency, which is size-exclusion of proteins from regions of stagnant flow. The latter is supported by recently published data on restricted protein diffusion in face-centered cubic silica colloidal crystals. Extremely low plate heights are enabled by use of submicrometer particles, and further improvement appears to be possible when the analyte size is on the order of 1% of the particle diameter or larger.

Obstructed diffusion in silica colloidal crystals.

The hindered diffusion in silica colloidal crystals was studied experimentally, both by fluorescence recovery after photobleaching and by measurement of ionic conductivity. Particle size was varied to include 120, 220, 470, and 1300 nm, and the porosities were determined by flow measurements. For fluorescein, the results showed that the obstruction factor, which is the ratio of the diffusion coefficients inside the media and in open solution, is equal to the porosity within experimental error. For proteins, the same conclusion is made after correction for size exclusion of the pores. The obstruction factors for these media are 2-fold lower than those measured for chromatographic media, 60% higher than theoretical predictions, and equal to what is assumed for electrophoretic sieving in random fibers.

Modeling of protein electrophoresis in silica colloidal crystals having brush layers of polyacrylamide.

Sieving of proteins in silica colloidal crystals of millimeter dimensions is characterized for particle diameters of nominally 350 and 500 nm, where the colloidal crystals are chemically modified with a brush layer of polyacrylamide. A model is developed that relates the reduced electrophoretic mobility to the experimentally measurable porosity. The model fits the data with no adjustable parameters for the case of silica colloidal crystals packed in capillaries, for which independent measurements of the pore radii were made from flow data. The model also fits the data for electrophoresis in a highly ordered colloidal crystal formed in a channel, where the unknown pore radius was used as a fitting parameter. Plate heights as small as 0.4 µm point to the potential for miniaturized separations. Band broadening increases as the pore radius approaches the protein radius, indicating that the main contribution to broadening is the spatial heterogeneity of the pore radius. The results quantitatively support the notion that sieving occurs for proteins in silica colloidal crystals, and facilitate design of new separations that would benefit from miniaturization.

Slip flow through colloidal crystals of varying particle diameter.

Slip flow of water through silica colloidal crystals was investigated experimentally for eight different particle diameters, which have hydraulic channel radii ranging from 15 to 800 nm. The particle surfaces were silylated to be low in energy, with a water contact angle of 83°, as determined for a silylated flat surface. Flow rates through centimeter lengths of colloidal crystal were measured using a commercial liquid chromatograph for accurate comparisons of water and toluene flow rates using pressure gradients as high as 10(10) Pa/m. Toluene exhibited no-slip Hagen-Poiseuille flow for all hydraulic channel radii. For water, the slip flow enhancement as a function of hydraulic channel radius was described well by the expected slip flow correction for Hagen-Poiseuille flow, and the data revealed a constant slip length of 63 ± 3 nm. A flow enhancement of 20 ± 2 was observed for the smallest hydraulic channel radius of 15 nm. The amount of slip flow was found to be independent of shear rate over a range of fluid velocities from 0.7 to 5.8 mm/s. The results support the applicability of the slip flow correction for channel radii as small as 15 nm. The work demonstrates that packed beds of submicrometer particles enable slip flow to be employed for high-volume flow rates.