Introduction of Octadecyl-Bonded Porous Particles in 3D-Printed Transparent Housings with Multiple Outlets.

Microfluidic devices for comprehensive three-dimensional spatial liquid chromatography will ultimately require a body of stationary phase with multiple in- and outlets. In the present work, 3D printing with a transparent polymer resin was used to create a simplified device that can be seen as a unit cell for an eventual three-dimensional separation system. Complete packing of the device with 5-µm C18 particles was achieved, with reasonable permeability. The packing process could be elegantly monitored from the pressure profile, which implies that optical transparency may not be required for future devices. The effluent flow was different for each of the four outlets of the device, but all flows were highly repeatable, suggesting that correction for flow-rate variations is possible. The investigation into flow patterns through the device was supported by computational-fluid-dynamics simulations. A proof-of-principle separation of four standard peptides is described, with mass-spectrometric detection for each of the four channels separately. Supplementary Information: The online version contains supplementary material available at 10.1007/s10337-022-04156-w.

Development of comprehensive two-dimensional low-flow liquid-chromatography setup coupled to high-resolution mass spectrometry for shotgun proteomics.

Bottom-up proteomics provides often small amounts of highly complex samples that cannot be analysed by direct mass spectrometry (MS). To gain a better insight in the sample composition, liquid chromatography (LC) and (comprehensive) two-dimensional liquid chromatography (2D-LC or LC × LC) can be coupled to the MS. Low-flow separations are attractive for HRMS analysis, but they tend to be lengthy. In this work, a low-flow, online, actively modulated LC × LC system, based on hydrophilic-interaction liquid chromatography (HILIC) in the first dimension and reversed-phase liquid chromatography (RPLC) in the second dimension, was developed to separate complex mixtures of peptides. Miniaturization permitted the analysis of small sample amounts (1-5 µg) and direct coupling with micro-ESI MS (1 µL min-1). All components were focused and automatically transferred from HILIC to RPLC using stationary-phase-assisted active modulation (C18 traps) to deal with solvent-incompatibility or dilution issues. Optimization of the setup was performed for the HILIC columns and the RPLC columns to provide a more efficient separation and higher identification rates than obtained using one-dimensional (1D) LC. A 60% increase in peak capacity was obtained with the 2D setup compared to a 1D-RPLC separation and a 17-34% increase in the number of proteins identified was achieved for the samples analysed (2D-yeast-8280 peptides and 2D-kidney tissue-8843 peptides), without increasing the analysis time (2 h).

Accurate modelling of the retention behaviour of peptides in gradient-elution hydrophilic interaction liquid chromatography.

The applicability of models to describe peptide retention in hydrophilic interaction liquid chromatography (HILIC) was investigated. A tryptic digest of bovine-serum-albumin (BSA) was used as a test sample. Several different models were considered, including adsorption, mixed-mode, exponential, quadratic and Neue-Kuss models. Gradient separations were performed on three different HILIC stationary-phases under three different mobile-phase conditions to obtain model parameters. Methods to track peaks for specific peptides across different chromatograms are shown to be essential. The optimal mobile-phase additive for the separation of BSA digest on each of the three columns was selected by considering the retention window, peak width and peak intensity with mass-spectrometric detection. The performance of the models was investigated using the Akaike information criterion (AIC) to measure the goodness-of-fit and evaluated using prediction errors. The F-test for regression was applied to support model selection. RPLC separations of the same sample were used to test the models. The adsorption model showed the best performance for all the HILIC columns investigated and the lowest prediction errors for two of the three columns. In most cases prediction errors were within 1%.

Peak-Tracking Algorithm for Use in Automated Interpretive Method-Development Tools in Liquid Chromatography.

A peak-tracking algorithm for chromatograms recorded using liquid chromatography and mass spectrometry was developed. Peaks are tracked across chromatograms using the spectrometric information, the statistical moments of the chromatographic peaks, and the relative retention. The algorithm can be applied to pair chromatographic peaks in two very different chromatograms, obtained for different samples using different methods. A fast version of the algorithm was specifically tailored to process chromatograms obtained during method development or optimization, where a few similar mobile-phase-composition gradients (same eluent components, but different ranges and programming rates) are applied to the same sample for the purpose of obtaining model parameters to describe the retention of sample components. Due to the relative similarity between chromatograms, time-saving preselection protocols can be used to locate a candidate peak in another chromatogram. The algorithm was applied to two different samples featuring isomers. The automatically tracked peaks and the resulting retention parameters generally yielded prediction errors of less than 1%.

Capillary HILIC-MS: A New Tool for Sensitive Top-Down Proteomics.

Recent progress in top-down proteomics has driven the demand for chromatographic methods compatible with mass spectrometry (MS) that can separate intact proteins. Hydrophilic interaction liquid chromatography (HILIC) has recently shown good potential for the characterization of glycoforms of intact proteins. In the present study, we demonstrate that HILIC can separate a wide range of proteins exhibiting orthogonal selectivity with respect to reversed-phase LC (RPLC). However, the application of HILIC to the analysis of low abundance proteins (e.g., in proteomics analysis) is hampered by low volume loadability, hindering down-scaling of the method to column diameters below 2.1 mm. Moreover, HILIC-MS sensitivity is decreased due to ion suppression from the trifluoroacetic acid (TFA) often used as the ion-pair agent to improve the selectivity and efficiency in the analysis of glycoproteins. Here, we introduce a capillary-based HILIC-MS method that overcomes these problems. Our method uses RPLC trap-columns to load and inject the sample, circumventing issues of protein solubility and volume loadability in capillary columns (200 µm ID). The low flow rates and use of a dopant gas in the electrospray interface improve protein-ionization efficiencies and reduce suppression by TFA. Overall, this allows the separation and detection of small protein quantities (down to 5 ng injected on column) as indicated by the analysis of a mixture of model proteins. The potential of the new capillary HILIC-MS is demonstrated by the analysis of a complex cell lysate.