Exploiting ion-mobility mass spectrometry for unraveling proteome complexity.

Ion mobility spectrometry-mass spectrometry (IMS-MS) is experiencing rapid growth in proteomic studies, driven by its enhancements in dynamic range and throughput, increasing the quantitation precision, and the depth of proteome coverage. The core principle of ion mobility spectrometry is to separate ions in an inert gas under the influence of an electric field based on differences in drift time. This minireview provides an introduction to IMS operation modes and a description of advantages and limitations is presented. Moreover, the principles of trapped IMS-MS (TIMS-MS), including parallel accumulation-serial fragmentation are discussed. Finally, emerging applications linked to TIMS focusing on sample throughput (in clinical proteomics) and sensitivity (single-cell proteomics) are reviewed, and the possibilities of intact protein analysis are discussed.

A protocol for fabrication of polymer monolithic capillary columns and tuning the morphology targeting high-resolution bioanalysis in gradient-elution liquid chromatography.

Polymer monolithic stationary phases are designed as a continuous interconnected globular material perfused by macropores. Like packed column, where separation efficiency is related to particle diameter, the efficiency of monoliths can be enhanced by tuning the size of both the microglobules and macropores. This protocol described the synthesis of poly(styrene-co-divinylbenzene) monolithic stationary phases in capillary column formats. Moreover, guidelines are provided to tune the macropore structure targeting high-throughput and high-resolution monolith chromatography. The versatility of these columns is exemplified by their ability to separate tryptic digests, intact proteins, and oligonucleotides under a variety of chromatographic conditions. The repeatability of the presented column fabrication process is demonstrated by the successful creation of 12 columns in three different column batches, as evidenced by the consistency of retention times (coefficients of variance [c.v.] = 0.9%), peak widths (c.v. = 4.7%), and column pressures (c.v. = 3.1%) across the batches.

Removal of Low Trace ppb-Level Perfluorooctanesulfonic Acid (PFOS) with ZIF-8 Coatings Involving Adsorbent Degradation.

For the first time, low trace-level removal of perfluorooctanesulfonic acid (PFOS), i.e., 20-500 µg/L (ppb), from aqueous solutions using zeolitic imidazolate framework-8 (ZIF-8)-coated copper sheet (ZIF-8@Cu) composite is reported here. In comparison with different commercial activated carbon (AC) and all-silica zeolites, the composite showed the highest removal rate of 98%, which remained consistent over a wide range of concentrations. Additionally, no adsorbent leaching from the composite was noticed, which eradicated pre-analysis steps such as filtration and centrifugation, unless needed for other adsorbents studied here. The composite displayed fast uptake with saturation reaching within 4 h, irrespective of the initial concentration. However, the morphological and structural characterization revealed surface degradation of ZIF-8 crystals, along with a decline in the crystal size. The adsorption of PFOS on ZIF-8 crystals was linked to chemisorption, as the surface degradation surges with an increase in PFOS concentration or with cyclic exposure at low concentrations. Methanol seemingly removed surface debris (partially), thus providing access to ZIF-8 beneath the surface debris. Overall, the findings demonstrate that at low trace ppb-level PFOS concentrations ZIF-8 can be considered as a possible candidate for PFOS removal, even though it suffers slow surface degradation, it also removes efficiently PFOS molecules from aqueous solutions.

Development of a generic ultra-high-pressure gradient liquid-chromatography method development protocol: The analysis of residual multi-class antibiotics in food products as a case study.

The present study discusses UHPLC method development allowing to establish ultra-high-resolution separations in gradient mode while operating at the kinetic performance limits, targeting the analysis of complex residual multi-class antibiotic samples in food products. The peak capacity and gradient occupation have been systematically assessed at different flow rates and gradient duration. The small particle size (1.5 µm core-shell particles) used in this study limits the mass-transfer contribution to band broadening when operating at high flow rate. As a result, for high-throughput analysis, high-pressure (1500 bar) operation leads to high resolving power where the gradient steepness dominates the peak capacity generation vs mass-transfer resistance. To reach the highest possible resolving power within a practically acceptable analysis time, one should use coupled-column systems at 1500 bar and adjust the gradient steepness correspondingly. Coupling four columns and applying a shallow gradient at 1500 bar led to a sample peak capacity of 379 in 140 min, allowing to resolve 71% of the analytes in a mixture composed of 61 milk antibiotics.

Assessing effects of ultra-high-pressure liquid chromatography instrument configuration on dispersion, system pressure, and retention.

This study involves the systematic assessment of the effects of system configuration on dispersion, pressure, and retention characteristics while operating a 1500 bar UHPLC system with 2.1 mm i.d. × 100 mm long columns packed with 1.5 µm core-shell particles in isocratic and gradient mode. Altering the system configuration by changing the i.d. of connection tubing and flow cells affects the elution time, dispersion characteristics, and the kinetic performance limits of the system. The gain in separation efficiency when decreasing tubing i.d. from 100 to 75 µm was found to contribute more to the decrease in separation impedance and the position of the kinetic performance curve than the loss in available column pressure induced by the narrower tubing. When applying steep gradients, characterized by gradient-to-column dead-time ratio < 7, optimizing instrument configuration leads to either a significant time gain factor of 3.9 without compromising peak capacity, or a gain in peak capacity with a gain factor of 1.3 while maintaining the analysis time constant. Due to the reduced fluidic volume of connection tubing of smaller i.d., a decrease in residence time is obtained. At the same time, an increase in k was observed due to a pressure-induced retention effect, and this effect is significant for late-eluting analytes.

Towards high peak capacity separations in normal pressure nanoflow liquid chromatography using meter long packed capillary columns.

Single shot proteomics is a promising approach to high throughput proteomics analysis. In this strategy, long capillary columns are needed to perform long and shallow gradients to achieve high peak capacity and good peak width for informative mass spectrometric detection. Herein, we report that meter long capillary columns, packed with 5 µm particulate material, can be facilely fabricated based on single particle fritting technology. The long columns could reliably generate high peak capacities of 800 in 10 h long gradients for protein digest separations. The operation was within the pressure range (40 MPa) of the most widely used normal pressure nanoLC systems. Due to the excellent life time (>100 injections) and inter-column performance consistency, the meter long capillary columns reported here should be of practical usefulness in single shot proteomics without the need for ultra-high pressure instrumentation.

A comparison study of in-column and on-column detection for electrochromatography.

Duplex capillary columns, the standard for electrochromatography using optical detection, consist of a packed and an open section. Normally, optical detection is performed in an on-column manner, i.e. at a point right after the packed section. It was deemed that band broadening may take place when an analyte band travels from the packed bed, through the frit and down to the open section. In this study, without using any sintering steps for fritting or window creation, robust packed capillary columns were prepared using transparent capillaries based on single particle fritting technology. The detection point could be easily shifted by simply sliding the transparent column against the ultraviolet (UV) beam. In this way, the band broadening effect was directly evaluated as a function of the detection point, which was positioned before or after the end frit. The consistent van Deemter curves recorded indicate that there was no efficiency difference between the positions investigated. The result proved that the significant band broadening effect previously observed via on-column detection should be caused by the sintered frit used, while the single particle frit made through a purely physical process did not lead to efficiency degradation. The conservative separation performance recorded at different positions around the column's end also suggests the applicability of on-line tandem detection strategy, e.g. UV followed by mass spectrometry (MS), on the same capillary column, which should be a promising approach to mining multiplex detection information from a single microseparation process.

Toward rapid preparation of capillary columns for electrochromatography use.

CEC is a high performance electrodriving liquid phase separation technique. It does not need complex and sophisticated high pressure instrumentation for nanoflow driving. This is attractive for parallel multicolumn analysis. To this end, high throughput methods for column preparation are needed to support the use of multiple columns. In this study, we directly used CEC mobile phase solution as the packing solvent, and realized rapid preparation of capillary columns based on a single particle fritting technology. The method presented high preparation throughput compared with other reported methods based on various fritting technologies. The single particle fritting approach promoted column preparation throughput to 1 column/h, including all the fritting, packing and conditioning steps. The rapidly prepared columns showed consistently high efficiency of up to 150 000 plates per meter, and usefulness in reversed phase CEC of neutral, charged and biomolecules. With standard peptides as the sample, excellent long term reproducibility (better than 0.8%RSD, ten days, for retention times) was observed.