Column-Only Band Broadening in a Porous Shell Radially Elongated Pillar Array Column.

In the quest for better performing separation media for liquid chromatography, micropillar array columns have received great interest over the past years. While previous research was mainly focused around micropillar array columns (µPACs) filled with cylindrical pillars, this contribution discusses µPACs with rectangular pillars, which, for the first time, have been anodized and hence carry a mesoporous shell. We report on a series of on-chip measurements of the band broadening and flow permeability in a µPAC with very wide radially elongated pillars (3·75 µm) and with an interpillar distance (2 µm) between that of the first (2.5 µm) and second generation (1.25 µm) of cylindrical µPACs. Because of the extreme flow path tortuosity, this type of µPAC can produce very large plate numbers over a short distance. Despite the relatively large interpillar distance, we obtain Hmin = 0.26 µm for a nearly unretained component (phase retention factor, k' ≈ 0.24) and Hmin = 0.79 µm for a retained component with k' ≈ 3. The kinetic performance in terms of separation impedance (Ei = 19) is considerably improved compared to cylindrical pillar µPACs (Ei in range 40-50) and is in excellent agreement with the theoretical value for an open tubular channel with a rectangular cross-section (Ei = 18). This shows that rectangular µPACs can be represented as a parallel bundle of interconnected open-tubular channels.

Exploration of cell state heterogeneity using single-cell proteomics through sensitivity-tailored data-independent acquisition.

Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain obscured with population-based techniques. The limited amount of material encapsulated in a single cell however, raises significant technical challenges to molecular profiling. Due to extensive optimization efforts, single-cell proteomics by Mass Spectrometry (scp-MS) has emerged as a powerful tool to facilitate proteome profiling from ultra-low amounts of input, although further development is needed to realize its full potential. To this end, we carry out comprehensive analysis of orbitrap-based data-independent acquisition (DIA) for limited material proteomics. Notably, we find a fundamental difference between optimal DIA methods for high- and low-load samples. We further improve our low-input DIA method by relying on high-resolution MS1 quantification, thus enhancing sensitivity by more efficiently utilizing available mass analyzer time. With our ultra-low input tailored DIA method, we are able to accommodate long injection times and high resolution, while keeping the scan cycle time low enough to ensure robust quantification. Finally, we demonstrate the capability of our approach by profiling mouse embryonic stem cell culture conditions, showcasing heterogeneity in global proteomes and highlighting distinct differences in key metabolic enzyme expression in distinct cell subclusters.

On-Chip Comparison of the Performance of First- and Second-Generation Micropillar Array Columns.

Because of its dimensions, the recently introduced micropillar array columns are most suited for high-efficiency liquid chromatography separations in proteomics. Unlike the packed bed columns and capillary-based column formats, the micropillar array concept still has significant room to progress in terms of the reduction of its characteristic size (i.e., pillar diameter and interpillar distance) to open the road to even higher-efficiency separations and their applications. We report here on the on-chip comparison between first-generation (Gen 1) and second-generation (Gen 2) micropillar array columns wherein the pillar and interpillar size have been halved. Because of the on-chip measurements, the observed plate heights H represent the fundamental band broadening, devoid of any extra-column band-broadening effects. The observed reduction of H with a factor of 2 around the uopt-velocity and with a factor of 4 in the C-term dominated regime of the van Deemter-curve is in full agreement with the theoretically expected gain. This shows the pillar and interpillar size reduction could be effectuated without affecting the theoretical separation potential of the micropillar arrays. Compared to Gen 1, Gen 2 offers a 4-fold reduction of the required analysis time around the optimal velocity and about a 16-fold reduction in the C-term-dominated range.

Ultrasensitive NanoLC-MS of Subnanogram Protein Samples Using Second Generation Micropillar Array LC Technology with Orbitrap Exploris 480 and FAIMS PRO.

In the light of the ongoing single-cell revolution, scientific disciplines are combining forces to retrieve as much relevant data as possible from trace amounts of biological material. For single-cell proteomics, this implies optimizing the entire workflow from initial cell isolation down to sample preparation, liquid chromatography (LC) separation, mass spectrometer (MS) data acquisition, and data analysis. To demonstrate the potential for single-cell and limited sample proteomics, we report on a series of benchmarking experiments where we combine LC separation on a new generation of micropillar array columns with state-of-the-art Orbitrap MS/MS detection and high-field asymmetric waveform ion mobility spectrometry (FAIMS). This dedicated limited sample column has a reduced cross section and micropillar dimensions that have been further downscaled (interpillar distance and pillar diameter by a factor of 2), resulting in improved chromatography at reduced void times. A dilution series of a HeLa tryptic digest (5-0.05 ng/µL) was used to explore the sensitivity that can be achieved. Comparative processing of the MS/MS data with Sequest HT, MS Amanda, Mascot, and SpectroMine pointed out the benefits of using Sequest HT together with INFERYS when analyzing sample amounts below 1 ng. Here, 2855 protein groups were identified from just 1 ng of HeLa tryptic digest hereby increasing detection sensitivity as compared to a previous contribution by a factor well above 10. By successfully identifying 1486 protein groups from as little as 250 pg of HeLa tryptic digest, we demonstrate outstanding sensitivity with great promise for use in limited sample proteomics workflows.

Evaluation of micropillar array columns for chromatographic separation of phosphorothioated oligonucleotides and their diastereomers.

Chromatographic analysis of therapeutic oligonucleotides is challenging due to the presence of closely related impurities, degradants or metabolites and due to the presence of phosphorothioate bonds, which introduce chiral centers. In the present study, ion pair reversed phase chromatography of oligonucleotides on micropillar array columns was investigated. Two commonly used mobile phase conditions were included. With 16.3 mM triethylamine and 400 mM hexafluoroisopropanol, the separation of 16-mer oligonucleotides differing in the number and positions of phosphorothioate linkages as well as some n-1 and n-2 truncations demonstrated complete suppression of diastereoselectivity. Although the positional phosphorothioate isomers evaluated could not be resolved, an increase in phosphorothioate bonds resulted in more retention. A therapeutic 19-mer RNA sequence with 2'-fluor and 2'-O-methyl modifications showed partial separation of some very close impurities. When using 15 mM triethyl ammonium acetate in the mobile phase, diastereomer selectivity was clearly observed for all analytes. The best result was obtained for the 19-mer RNA therapeutic mimic with four phosphorothioate bonds, since all 16 theoretical diastereomers were clearly observed under the conditions tested. A limited benchmark exercise demonstrated the improved capability of the new micropillar array columns. Therefore, these columns can be positioned as a valuable alternative when challenging oligonucleotide separations are expected.

Improved Sensitivity in Low-Input Proteomics Using Micropillar Array-Based Chromatography.

Capitalizing on the massive increase in sample concentrations which are produced by extremely low elution volumes, nanoliquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS) is currently one of the most sensitive analytical technologies for the comprehensive characterization of complex protein samples. However, despite tremendous technological improvements made in the production and the packing of monodisperse spherical particles for nanoflow high-pressure liquid chromatography (HPLC), current state-of-the-art systems still suffer from limits in operation at the maximum potential of the technology. With the recent introduction of the µPAC system, which provides perfectly ordered micropillar array based chromatographic support materials, completely new chromatographic concepts for optimization toward the needs of ultrasensitive proteomics become available. Here we report on a series of benchmarking experiments comparing the performance of a commercially available 50 cm micropillar array column to a widely used nanoflow HPLC column for the proteomics analysis of 10 ng of tryptic HeLa cell digest. Comparative analysis of LC-MS/MS-data corroborated that micropillar array cartridges provide outstanding chromatographic performance, excellent retention time stability, and increased sensitivity in the analysis of low-input proteomics samples and thus repeatedly yielded almost twice as many unique peptide and unique protein group identifications when compared to conventional nanoflow HPLC columns.

Suppression of the sidewall effect in pillar array columns with radially elongated pillars.

An important bottleneck of pillar array columns designed for liquid chromatography is that small deviations of the target 'magical distance' at the sidewall region leads to detrimental sidewall effects, due to local differences of linear velocities at the sidewall region versus other locations in the pillar bed. In the present study, we demonstrate that a lateral elongation of the pillar significantly increases the tolerance for offsets of the magical distance. By shifting the sidewall distance 600 nm for 2 pillar aspect ratio (AR) designs (AR=3 and 9), only minor sidewall effects on the measured plate heights could be observed for the AR=9 columns, while the plate height was roughly doubled when using the wrong versus the correct sidewall distance for the AR=3 columns. Technologically, this constitutes a huge advantage because small deviations (order of 100 nm) between the set and the finally achieved value for the inter-pillar distance are very common using mid-UV lithography based etching processes.

Integration of uniform porous shell layers in very long pillar array columns using electrochemical anodization for liquid chromatography.

Electrochemical anodization has been applied to grow porous shell layers of 300 nm (30 nm pores) in 5 µm diameter pillar array columns (PACs) with a spacing of 2.5 µm. Using turn structures preceded and followed by the flow distributor structures recently introduced by our group and filled with radially elongated pillars, columns with quasi unlimited channel lengths could be conceived. The uniformity of the porous PAC was assessed by determining local plate heights along the channel, which appeared to be constant. Minimal (absolute) plate heights (H) between 4 and 6 µm were obtained at optimal flow rates when imposing increasing retention factors. Upon measuring the surface area involved in chromatographic retention as an indicator of the available surface area, an increase in the surface area by a factor of about 30 compared to that of non-anodized pillars was found. On reconfiguring a commercial HPLC instrument to enable on-chip injections, 90% of the performance (expressed in theoretical plates) could be maintained for a 1 m column, while for a 25 cm column severe losses were still observed. As the corresponding pressure drop for optimal operation of retained components is on the order of 10 bar per m only, portable and cheaper HPLC devices with high efficiencies become realistically conceivable.

On the advantages of radially elongated structures in microchip-based liquid chromatography.

We report on the possibility to realize submicrometer plate heights using chromatographic pillar array columns filled with radially elongated diamond-shaped pillars, even when using a relatively large interpillar distance (2.5 µm) and axial pillar width (5 µm). It is demonstrated that the use of high aspect ratio radially elongated pillars which are 15 times wider in the radial than in the axial direction can lead to a fivefold reduction of the minimal plate height compared to beds filled with pillars with a similar interpillar distance but with an aspect ratio around unity (cylinders and diamonds).This increase in performance can be attributed to a decrease in longitudinal dispersion, reflected by a reduction of the B-term by a factor of about 25. Experiments were conducted at room temperature, as well as at elevated temperature (70 °C), where the B-term band broadening is known to be more critical. The main advantage of radially elongated pillar beds is that they enable a drastic reduction of the footprint of pillar array columns, allowing design of very long channels with a minimum of turns. Under retained conditions, a four-component laser dye mixture could be separated over a distance of only 1.5 mm.

Hydrodynamic chromatography separations in micro- and nanopillar arrays produced using deep-UV lithography.

We report on a series of hydrodynamic chromatography separations conducted in micropillar array columns with an interpillar distance spacing of, respectively, 1.00, 0.70, and 0.47 µm. The columns have been produced using state-of-the-art deep-UV lithography and deep reactive ion etching techniques. Despite the fact that the efficiency was smaller than theoretically possible (due to fabrication limitations and significant injection and detection band broadening), it was nevertheless possible to separate mixtures of fluorescein isothiocyanate (used as the t(0) -marker) and 20- and 40-nm polystyrene beads. With the smallest interpillar distance, a resolution of R(s) = 0.5 between the 20- and 40-nm particles could be obtained in 90s over a column length of 4 cm. The selectivity obtained in the pillar array columns was found to be very similar to that observed in packed-bed columns. By detecting the fluorescent signals in a 90-µm-deep detection groove at the end of the column, the signal-to-noise ratio could be enhanced up to 150 times.

Separations using a porous-shell pillar array column on a capillary LC instrument.

We investigated the achievable separation performance of a 9-cm-long and 1-mm-wide pillar array channel (volume = 0.6 µL) containing 5 µm diameter Si pillars (spacing 2.5 µm) cladded with a mesoporous silica layer with a thickness of 300 nm, when this channel is directly interfaced to a capillary LC instrument. The chip has a small footprint of only 4 cm × 4 mm and the channel consists of three lanes that are each 3 cm long and that are interconnected using low dispersion turns consisting of a narrow U-turn (10 µm), proceded and preceded by a diverging flow distributor. Measuring the band broadening within a single lane and comparing it to the total channel band broadening, the additional band broadening of the turns can be estimated to be of the order of 0.5 µm around the minimum of the van Deemter curve, and around some 1 µm (nonretained species) and 2 µm (retained species) in the C-term dominated regime. The overall performance (chip + instrument) was evaluated by conducting gradient elution separations of digests of cytochrome c and bovine serum albumin. Peak capacities up to 150 could be demonstrated, nearly completely independent of the flow rate.

Impact of the limitations of state-of-the-art micro-fabrication processes on the performance of pillar array columns for liquid chromatography.

We report on the practical limitations of the current state-of-the-art in micro-fabrication technology to produce the small pillar sizes that are needed to obtain high efficiency pillar array columns. For this purpose, nine channels with a different pillar diameter, ranging from 5 to 0.5 µm were fabricated using state-of the-art deep-UV lithography and deep reactive ion etching (DRIE) etching technology. The obtained results strongly deviated from the theoretically expected trend, wherein the minimal plate height (H(min)) would reduce linearly with the pillar diameter. The minimal plate height decreases from 1.7 to 1.2 µm when going from 4.80 to 3.81 µm diameter pillars, but as the dimensions are further reduced, the minimal plate heights rise again to values around 2 µm. The smallest pillar diameter even produced the worst minimal plate height (4 µm). An in-depth scanning electron microscopy (SEM) inspection of the different channels clearly reveals that these findings can be attributed to the micro-fabrication limitations that are inevitably encountered when exploring the limits of deep-UV lithography and DRIE etching processes. When the target dimensions of the design approach the etching resolution limits, the band broadening increases in a strongly non-linear way with the decreased pillar dimensions. This highly non-linear relationship can be understood from first principles: when the machining error is of the order of 100-200 nm and when the target design size for the inter-pillar distance is of the order of 250 nm, this inevitably leads to pores that will range in size between 50 and 450 nm that we want to highlight with our paper highly non-linear relationship. This highly non-linear relationship can be understood from first principles: when the machining error is of the order of 100-200 nm and when the target design size for the inter-pillar distance is of the order of 250 nm, this inevitably leads to pores that will range in size between 50 and 450 nm.

Realization of 1 × 10(6) theoretical plates in liquid chromatography using very long pillar array columns.

We report on the possibility to achieve ultra high efficiencies (order of 1 million theoretical plates) in liquid chromatography in a relatively short time of 20 min (elution time of unretained marker). This was achieved using a micropillar array column with optimized pillar diameter (5 µm) and interpillar distance (2.5 µm) to operate close to the Knox and Saleem limit of micropillar array columns in the region of the 1 million theoretical plate mark under the prevailing pressure restriction (350 bar in the present study). The obtained efficiency was slightly affected (some 15 to 20% around the optimal flow rate) by the turns that were inevitably needed to arrange a 3 m long column on a 4 in. silicon wafer.

Micron-sized pillars for ion-pair reversed-phase DNA separations.

In the present paper, the feasibility to construct micron-sized silicon pillar channels to be used in HPLC is studied. For this, a channel with flow-through pores of 1 µm and with critical sidewall dimensions below 1 µm was constructed using advanced deep-UV lithographic equipment. Integrating a 3-nL injection system on the chip directly in front of the separation channel and using elongated distribution structures, a very controlled and high aspect ratio sample definition across the relatively wide separation channel was accomplished. The system was evaluated in isocratic ion-pair RP mode, allowing the separation of a mixture of two components with, respectively, 300 and 400 base pairs in 5 s only.

Hydrodynamic chromatography of polystyrene microparticles in micropillar array columns.

We report on the possibility to perform HDC in micropillar array columns and the potential advantages of such a system. The HDC performance of a pillar array column with pillar diameter = 5 microm and an interpillar distance of 2.5 microm has been characterized using both a low MW tracer (FITC) and differently sized polystyrene bead samples (100, 200 and 500 nm). The reduced plate height curves that were obtained for the different investigated markers all overlapped very well, and attained a minimum value of about h(min)=0.3 (reduction based on the pillar diameter), corresponding to 1.6 microm in absolute value and giving good prospects for high efficiency separations. The obtained reduced retention time values were in fair agreement with that predicted by the Di Marzio and Guttman model for a flow between flat plates, using the minimal interpillar distance as characteristic interplate distance.

Design and evaluation of flow distributors for microfabricated pillar array columns.

Five different flow distributors have been compared as a function of the flow rate for their ability to distribute small sample volumes over the entire width of flat rectangular microfabricated pillar array columns. The investigated designs can be divided in two major categories: (1) bifurcating, radially non-interconnecting distributors and (2) radially interconnecting distributors consisting of diamond-shaped pillars, elongated in the direction perpendicular to the flow, providing a high ratio of radial permeability over axial permeability. The quality of the flow distribution was evaluated experimentally by injecting equal volumes of fluorescent tracer into each of the tested designs and calculating the obtained peak variances using the method of moments. Purely bifurcating distributors perform less well than the best possible radially interconnected distributors, because the former inevitably require the use of wide open channels (d > 10 microm), wherein a lot of band broadening can occur. By doubling the aspect ratio of the radially stretched pillars from 5 to 10, the measured peak variance drops to 1/8 of the original value. The best results were obtained with a distributor in which the flow is distributed by a bed of anisotropic pillars with an aspect ratio of 10, but our results indicate that a substantial improvement can still be made by increasing the aspect ratio and adding gradually diverging sidewalls to the inlet.

Visualization and quantification of the onset and the extent of viscous fingering in micro-pillar array columns.

New experimental data of the viscous fingering (VF) process have been generated by studying the VF process in perfectly ordered pillar array columns instead of in the traditionally employed packed bed columns. A detailed quantitative analysis of the contribution of VF to the observed band broadening could be made by following the injected species bands using a fluorescence microscope equipped with a CCD-camera. For a viscosity contrast of 0.16 cP, a plate height increase of about 1 microm can be observed, while for a contrast of respectively 0.5 cP and 1 cP, additional plate height contributions of the order of 5-20 microm were observed. Citing these values is however futile without noting that they also depend extremely strongly on the injection volume of injected sample. It was found that, for a given viscosity contrast of 0.314 cP, the maximal plate height increase varied between 0.5 microm and 18 microm if the injection volume was varied between 3.0 nl and 32.7 nl. These values furthermore also strongly vary with the distance along the column axis.

Prolonged somatostatin therapy may cause down-regulation of SSTR-like GPCRs on Schistosoma mansoni.

BACKGROUND & OBJECTIVES: Chemotherapy with praziquantel remains the only control measure to Schistosoma mansoni infections to date. The neuropeptide hormone somatostatin gives relief from gastrointestinal disturbances, liverpathology, and reduces egg production in S. mansoni infected mice, suggesting an interaction of somatostatin with the parasite rather than with the host alone. Using antibodies directed to epitopes of the seven somatostatin transmembrane receptors (SSTRs), the presence of SSTRs (or proteins that contain these epitopes) was shown on both worm- and egg-stages of S. mansoni. The present study was undertaken to investigate whether SSTRs on S. mansoni displayed homo/heterodimerisation properties as well as agonist induced down-regulation. RESULTS: Somatostatin therapy was effective after two days of treatment with no further reduction in pathology after five days of therapy. Immunohistochemistry performed on parasite sections showed reactivity of the anti-SSTR antibodies to the tegument and internal parts of adult S. mansoni worms. SDS-PAGE-Western blotting identified protein bands of 70-100 and 200-250 kDa molecular weight. Upon carboxymethylation of the sulfhydryl groups of proteins in the worm lysate, a reduction in density of the protein band at 200-250 kDa and an increase in density of the protein band at 70-100 kDa were noted. This suggested that a substantial amount of the proteins detected on the blot are present as a homo/heterodimer. A protein microarray was used to investigate whether somatostatin therapy induced receptor down- or up-regulation on the adult worm of S. mansoni. Slides spotted with primary anti-SSTR antibody were exposed to lysates of worms collected from infected C3H mice that received none, two days or five days somatostatin treatment, followed by a secondary anti-SSTR antibody coupled to a fluorophore. Comparison of the different samples in terms of parasite dilution till when the fluorescence was detectable, and the fluorescence intensity, proved that the proteins detected in the parasite worm have been down-regulated after five days of somatostatin treatment. CONCLUSION: SSTR-like GPCRs are being expressed by adult S. mansoni worms and extended somatostatin treatment may cause down-regulation of these receptors, thus reducing the therapeutic capacities of this neuropeptide. However, the presence of SSTRs on S. mansoni has not yet been proven on a genetic basis. Cross-reactivity of anti-SSTR antibodies with other G-protein coupled receptors (GPCR) thus cannot yet be excluded.