Thermo Scientific µPAC Neo HPLC Columns
by Paul Jacobs, PhD, R&D Director Microfluidic Separation Solutions, and Shanhua Lin, PhD, Senior Director of Research and Development, Thermo Fisher Scientific
Precision medicine is fast emerging as the next wave of medical innovation. Today, many patients still experience a one-size-fits-all approach when it comes to treatment of symptoms or prevention of diseases, which often overlooks the biological differences among us. But scientists are looking to a combination of “omics” studies, including genomics, proteomics, metabolomics, metagenomics, phenomics and transcriptomics, to provide deeper insights into the inner workings of health and disease and to potentially inform clinical decisions for more individualized care.
Technology has advanced tremendously in recent years and is still evolving to help scientists discover new information quicker and more efficiently. For researchers who study proteomics, which can be notoriously time consuming and complex, workflows that provide faster throughput, higher sensitivity and deeper coverage, even at single cell level, allow scientists to gather more valuable information each time a sample is processed. But more importantly, reproducibility ensures that the discoveries are meaningful. Micromachined high performance liquid chromatography (HPLC) columns, also referred to as micro pillar array columns (µPAC) enable scientists to consistently replicate sample runs in their labs. A new version of such columns now also allows scientists to achieve this at high throughput.
Improving on the basics of chromatography to see more details
HPLC columns are used to separate the components of a sample for analysis. Many different types of HPLC columns exist. They can be of different size, material or contain different chemistries. Choosing which type of column should be used will depend on the samples that will be run through it. In proteomics, low-flow columns need to be used to avoid excessive sample dilution and maximize sensitivity. Recently, a new approach of producing low-flow HPLC columns has been introduced. Traditionally columns have been manufactured by packing slurries of microparticles into a tube or capillary. For the low flow columns that are needed for proteomics, this process impacts the efficiency of the chromatographic column.
In contrast, micro pillar array columns (µPAC) are a newer type of columns of which the backbone of the stationary phase is truly designed, and manufacturing is based on replicating this design to result in perfectly ordered flow paths. The manufacturing starts from high purity silicon wafers, using state-of-the-art microtechnology, similar to how integrated circuits are made in a smartphone or a computer chip, for the benefit of making perfectly controlled microfluidic chromatographic separation devices. Using this approach has a number of beneficial consequences for the user:
- The ordered flow path of the column decreases the risk of sample dispersion inside the column, which will result in more distinguishable, less diluted chromatographic peaks, hence increasing sensitivity.
- Because of the tightly controlled spatial positioning of the freestanding pillars, these micro pillar array columns can be operated at moderate pressures, allowing for longer flow paths that increase separation capacity, whereas traditional packed bed columns are facing the limits of practically achievable instrument pressure1,2,3.
- The freestanding nature of the pillars, together with the monolithic solid character of the backbone, renders the columns extremely stable and robust, and increases column longevity4.
- The open structure results in low carry-over, making these columns attractive for low sample input and single cell work5.
- The silicon wafer-based manufacturing process results in the column-to-column reproducibility that is required to generate high-quality and consistent data each and every run and at all sites.
The combination of these unique characteristics creates the opportunity for the micro pillar array columns to bring this application to precision medicine research because of the consistency of the data and the quality of the experiments that can be done.
Achieving higher throughput, greater efficiency
These days, researchers are looking to run more samples each day, as equipment time can add up. The more samples that can be run in a day, the more cost effective it is to screen – especially large sample cohorts. To process these large sample groups, scientists need tools that are going to offer high throughput and greater efficiency throughout the workflow. From sample prep to columns and liquid chromatography to mass spectrometry and data interpretation, choosing technologies that work together to ease the process and limit human error are going to offer the greatest benefit to researchers.
New columns allow for high separation performance in a short time with high reproducibility, which makes it more appealing to scientists who are working toward precision medicine because of the consistent high-quality data and high throughput in sample analysis.
Moving proteomics into the clinic
As LC-MS-based proteomic research advances toward clinical and translational research applications, efficient workflows and data reproducibility are crucial for the future of precision medicine to become a reality. When we think about the future, where busy clinicians could be running multiple patient samples to look for relevant biomarkers in real-time, it’s even more important to consider the technology’s capability to produce consistent data each time.
While columns are only a part of the workflow, a system is only as good as the sum of its parts. It is essential that all touchpoints in the complete proteomics workflow offer high throughput screening and excellent stability for quantification quality that’s reproducible and allows for continuous data generation. It’s with the complete, robust workflows that we will see advancements in medicine toward a more personalized approach to healthcare, and columns are a critical component of the workflow that are not to be overlooked. With the introduction of microfluidic chromatography columns researchers have an important new option in their toolbox.
References
1. Jennifer E. Van Eyk et al., High Field Asymmetric Waveform Ion Mobility Spectrometry: Practical Alternative for Cardiac Proteome Sample Processing, J. Proteome Res. 2023, 22, 2124 – 2130.
2. van Leeuwen et al., The salivary proteome in relation to oral mucositis in autologous hematopoietic stem cell transplantation recipients: a labelled and label-free proteomics approach, BMC Oral Health 2023, 23:460
3. Lilian R. Heil et al., Evaluating the Performance of the Astral Mass Analyzer for Quantitative Proteomics Using Data Independent Acquisition, https://doi.org/10.1101/2023.06.03.543570.
4. Daniel Hornburg et al., Enhanced competitive protein exchange at the nano-bio interface enables ultra-deep coverage of the human plasma proteome, https://doi.org/10.1101/2022.01.08.475439.
5. Erwin M. Schoof et al., Evaluating the capabilities of the Astral mass analyzer for single-cell proteomics, https://doi.org/10.1101/2023.06.06.543943.