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Streamlining LC-MS Measurements for Proteomics Research

LOW-FLOW LC COLUMNS FACILITATE INCREASED SENSITIVITY AND HIGH RESOLUTIONS FOR LC-MS MEASUREMENTS


three experts

photo provided by thermo fisher scientific

Robert van Ling is currently the Low-flow HPLC Column product manager at Thermo Fisher Scientific. Robert started his career in the mid-90s in nano- and capillary LC at LC Packings, one of the leading companies in this then-young analytical field. Since then, he has held several roles in marketing and sales support for the separation and characterization of biomolecules and most recently was part of the introduction team for the Thermo Scientific™ µPAC™ HPLC columns.

Dr. Jeff Op de Beeck is currently Staff Scientist at Thermo Fisher Scientific’s R&D facility in Ghent where micropillar array technology is developed and produced. His work under the supervision of Prof. Gert De Smet and Prof. Wim De Malsche formed the basis for the current micro–Pillar Array Column, which is branded by Thermo Fisher Scientific as the µPAC™. He holds a Master’s degree in Biomedical Sciences from the University of Antwerp and a Ph.D. in Chemical Engineering from the University of Brussels.

Paul Jacobs is R&D Director at MSS (Microfluidic Separation Solutions) part of Thermo Fisher Scientific in Ghent, Belgium. Paul holds degrees in engineering in Physical Electronics, Biomedical Engineering, and a Ph.D. in Applied Sciences (Biosensors Development) from the University of Leuven, Belgium, which he complemented with several management training programs. Paul has accumulated extensive expertise in a wide variety of life sciences and medical device technologies, ranging from biosensors to in vitro diagnostic assays, as a research engineer and program manager in a few international companies as well as for start-ups. Paul was co-founder of PharmaFluidics.

Q: Why is LC-MS crucial to bottom-up and top-down proteomics research, and what does Thermo Fisher Scientific have to offer in this aspect? 

RL: In either top-down or bottom-up proteomics, mass spectrometry (MS) is the key technology. In combination with liquid chromatography (LC), MS provides high-efficient separations alongside sensitive detection as well as extensive identification and quantification of the proteins composing a highly complex sample or mixture. Thermo Scientific’s™ Orbitrap™ MS plays a crucial role in LC-MS top-down and bottom-up proteomics alongside various new innovations focusing specifically on low flow chromatography. These innovations include the Thermo Scientific™ Vanquish™ Neo, an optimized ultra-high-performance liquid chromatography ((UHPLC)) system, and the recently introduced μPAC HPLC columns. Together with the Orbitrap, they enable extremely high peak capacities and resolution separations at nano-LC flow rates ranging between 200 to 750 nanoliters per minute. Furthermore, the Thermo Scientific™ EASY-Spray™ PepMap™ Neo UHPLC columns serve as complementary counterparts to complete the bottom-up proteomics LC column portfolio.

Q: Compared to analytical flow LC-MS, what do low-flow columns offer for proteomics research, and what sets them apart?

JO: Low-flow LC columns facilitate increased sensitivity that can be achieved by moving toward lower flow rates. This is due to the electrospray ionization process, which tends to be much more effective at nano flow rates approaching several nanoliters per minute and up to 300 or 500 nanoliters per minute. This will bring increased ionization efficiency and detection sensitivity which is needed to identify and quantify low abundant biomarkers from precious biological samples. The combination of µPAC HPLC columns with the newly introduced Vanquish Neo system give excellent results by combining precise gradient formation at nano-LC flow rates with high resolution microchip-based LC separation with a user experience that makes it easier to use and optimize.

Q: What information can low-flow LC-MS extract and how is this facilitated by the advanced HPLC column solutions?

JO: In bottom-up proteomics, a digested cell lysate sample typically contains tens of thousands of peptides that come from a vast population of proteins, and these are often present over a huge dynamic concentration range. Low abundant proteins can have an important biological role, and therefore it is highly desired to have a method that allows identifying and quantifying them. This is where nano-LC comes in the picture. Apart from the ionization efficiency achieved using very narrow columns and low flow rates, nano-LC ensures that samples get less diluted and thus maximizing the signal response in MS measurements. The use of μPAC based LC column formats also allows operating optimized and longer separation channels at lower pressures, which further amplifies the resolution and peak capacity. These are also the reasons behind why the field has evolved into using nano-LC as the method of choice in proteomics, backed by the last decade of innovation in system hardware.

Q: What challenges had to be addressed in the development of these solutions and what remains unresolved?

PJ: The driving factor behind these solutions was to meet user demands for higher resolution measurements with increasing peak capacities and narrowing peaks. This was facilitated through the μPAC HPLC columns that eliminate eddy dispersion to increase separation resolution and combine relatively high loading and equilibration flow to reduce the overhead of these non-productive cycles in the analysis. This allows the gradient analysis to be run at the optimal flow rate for the nano-spray and thereby achieve the highest sensitivity. Most importantly, µPAC allows this to be done with high reliability. The μPAC HPLC columns are made out of single monolithic silicon blocks (wafers) with the pillars forming the backbone of the stationary phase extending from the bottom of the microchannel with no loose particles or frits. This makes them extremely stable and robust, providing for longevity and high retention time stability. As the μPAC HPLC columns are manufactured using lithography and micromachining, they are also highly reproducible and easily replaceable.