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INTRODUCTION
IonOpticks’ Aurora Series packed emitter columns feature our revolutionary nanoZero® fitting, allowing for simplified plug-and-play high-throughput proteomics. When coupled with cutting-edge mass spectrometry system, Aurora Series columns demonstrate exceptional performance. In particular, the combination between IonOpticks’ Aurora Frontier 60 cm x 75 μm XT columns and Thermo Fisher Scientific’s Orbitrap Exploris 480 mass spectrometer creates a powerful analytical platform, enabling in-depth analysis in proteomics research. To demonstrate the capabilities of this system, experiments were conducted using 3 replicates across different gradient lengths (30 min and 60 min), allowing for an evaluation of the column’s performance under various analytical conditions.
Maximise identifications of peptides and proteins
IonOpticks’ Aurora Frontier 60 cm x 75 μm XT columns offer exceptional performance in proteomics applications. These results demonstrate the advantages of using longer columns that deliver high-resolution separations, allowing researchers to gain deeper proteome coverage in their studies.
(A) UNIQUE PROTEIN GROUPS
(B) UNIQUE PEPTIDES
Figure 1: (A) Average protein group identifications across different gradients. (B) Average unique peptide identifications across different gradients. A HeLa tryptic digest (200 ng) was separated on Aurora Frontier 60 cm x 75 μm XT column. 3 replicates were run per condition. Samples were run on Thermo Fisher Scientic’s Vanquish Neo LC and Orbitrap Exploris 480 mass spectrometer with FAIMS Pro Duo. FAIMS CVs of -45V for 30 min gradient and -45/-65V for 60 min gradient were used. Data analysis was performed using Spectronaut 18 (Biognosys AG).
Narrow peak widths
The Aurora Frontier 60 cm x 75 μm XT column shows exceptional chromatographic performance by delivering narrow peak widths across varying gradient times. Peak widths (FWHM) average 3.0 seconds for 30 minute gradients and 5.5 seconds for 60 minute gradients.
Figure 2: Average full width at half maximum (FWHM) for all identified peptides from Hela Tryptic Digest injections (200 ng) on an Aurora Frontier 60 cm x 75 μm XT column. 3 replicates were run per condition. Each point represents the average FWHM for an individual replicate. Samples were run on Thermo Fisher Scientic’s Vanquish Neo LC and Orbitrap Exploris 480 mass spectrometer with FAIMS Pro Duo. FAIMS CVs of -45V for 30 min gradient and -45/-65V for 60 min gradient were used. Data analysis was performed using Spectronaut 18 (Biognosys AG).
FIG 2 AVERAGE FULL-WIDTH HALF-MAX
FIG 3 RETENTION TIME FOR SELECTED PEPTIDES
Stable retention times ensure confidence in results
The Aurora Frontier columns consistently show exceptional retention time stability over multiple runs for both 30 minute and 60 minute gradients. This high level of reproducibility is crucial in proteomics, as it ensures dependable peptide identification across various experimental conditions.
Figure 3: Stable peptide retention times across all runs. 6 peptides were selected and their retention time assessed across all runs from Hela Tryptic Digest injections (200 ng) on an Aurora Frontier 60 cm x 75 μm XT column. Samples were run on Thermo Fisher Scientic’s Vanquish Neo LC and Orbitrap Exploris 480 mass spectrometer with FAIMS Pro Duo. FAIMS CVs of -45V for 30 min gradient and -45/-65V for 60 min gradient were used. Data analysis was performed using DIA-NN software (Version 1.9).
Low CVs
Analyzing protein intensity reproducibility using the coefficient of variation (CV) reveals the exceptional consistency of the Aurora Frontier columns across various gradient lengths. Both 30 minute and 60 minute gradients shows low median CV values: 4.06% and 3.27% respectively. These outcomes highlight the robustness of the Aurora Frontier columns in delivering consistent performance.
Figure 4: Low CVs across different gradients. Violin plot of the coefficient of variation for all protein intensities identified across all replicates from HeLa Tryptic Digest injections (200 ng) on an Aurora Frontier 60 cm x 75 μm XT column. 3 replicates were run per condition. Samples were run on Thermo Fisher Scientic’s Vanquish Neo LC and Orbitrap Exploris 480 mass spectrometer with FAIMS Pro Duo. FAIMS CVs of -45V for 30 min gradient and -45/-65V for 60 min gradient were used. Data analysis was performed using Spectronaut 18 (Biognosys AG).
FIG 4 COEFFICIENT OF VARIATION (CV %)
CONCLUSION
The integration of IonOpticks’ Aurora Series columns with Thermo Fisher Scientific’s Vanquish Neo LC and Orbitrap Exploris 480 mass spectrometer achieves maximum protein and peptide identifications. This powerful combination enhances analytical capabilities, providing robust and precise results in proteomics research. By uniting these advanced technologies, researchers gain a reliable and precise platform for conducting cutting-edge proteomics studies, unlocking deeper insights into complex samples.
About IonOpticks
IonOpticks produces high-performance chromatography solutions for the global research community. We specialise in the development and manufacture of columns for analytical applications in liquid chromatography with mass spectrometry (LC-MS) and high-end proteomics. Our highly reproducible methods provide a unique ability to enhance the sensitivity of mass spectrometry sample analysis, enabling scientists and clinicians to discover more from their samples. Our team are experts in a broad array of LC-MS platform technologies and are driven by the need to improve chromatographic performance in order to achieve data quality and deep proteome coverage on a whole new scale.
Further Reading
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