Find us at HUPO 2024 in Dresden: Authors: Vincent Albrecht 1, Tim Heymann 1, Greta Briedyté 2, Johannes Bruno Müller-Reif 1.
Affiliations: 1 Max Planck Institute of Biochemistry, 2 IonOpticks Pty Ltd.
Introduction
Robust performance across a large patient plasma cohort analysis
IonOpticks’ Aurora Rapid 8×150 packed emitter columns feature our revolutionary nanoZero® fitting, allowing for simplified plug-and-play high-throughput proteomics. These columns are capable of analyzing from 50 to more than 100 samples per day while delivering class-leading peptide and protein identifications.
The Aurora Rapid 8×150 columns were used to analyze a large patient plasma cohort on an Evosep One and Thermo Fisher Orbitrap Astral. Weekly quality control (QC) of the LC-MS performance and reproducibility was performed by injection of 200ng HeLa tryptic peptides using a 60 SPD method. Plasma samples were analyzed using a
100 SPD method with approximately 700 plasma samples run between QC injections.
High-throughput analysis of peptides and proteins:
HeLa cell tryptic peptides, used for LC-MS QC, were injected and separated using a 60 samples per day method on an Evosep One. This method repeatedly identified over 9,300 proteins per run, with an average of 100,000 unique peptide identifications.
Figure 1: Protein identifications across different columns. A HeLa tryptic digest (200 ng) was separated on an Aurora Rapid 8 cm x 150 μm column on a 60SPD method using Evosep One and Thermo Fisher Orbitrap Astral Mass Spectrometer.
Figure 2: Unique peptide identifications across different columns. A HeLa tryptic digest (200 ng) was separated on an Aurora Rapid 8 cm x 150 μm column on a 60SPD method using Evosep One and Thermo Fisher Orbitrap Astral Mass Spectrometer.
High reproducibility across multiple columns
To assess the reproducibility of the protein quantification across multiple QC runs and columns, protein intensities were used to calculate a coefficient of variation (CV) and Pearson correlation matrix, revealing uniform performance with low CV values. Pearson correlation matrix visualization further supported this, showing high correlations between runs (mean Pearson correlation coefficient: 0.98).
Figure 3: Low CVs across different columns: Violin plot of coefficient of variation for
all identified protein intensities from HeLa tryptic digest QC injections on Aurora Rapid
8 cm x 150 μm columns. Samples were run on an Evosep One and Thermo Fisher
Orbitrap Astral Mass Spectrometer. Dashed line represents the median value for each
column.
Figure 4: A Pearson correlation matrix of all quantified protein intensities from the 257 QC injections was calculated.
Narrow peak widths
The below plot displays the full width at half maximum (FWHM) values across 257 QC runs, revealing consistently narrow peak widths. This consistent performance indicates good column quality and reproducibility, important for large patient cohort analyses.
Figure 5: Peak widths across all runs: average full width at half maximum (FWHM) for all identified peptides from Hela Tryptic Digest injections on an Aurora Rapid 8 cm x 150 μm columns. Samples were run on a Evosep One and Thermo Fisher Orbitrap Astral Mass Spectrometer.
Stable retention times ensure confidence in results
The analysis of retention time reproducibility is detailed through a scatter plot and histogram, focusing on the coefficient of variation for precursor retention times. The scatter plot demonstrates a high level of consistency, with most data points clustering below a CV of 6% and a median CV at 2.52%, showing stable retention times across precursors. Additionally, a four-month experiment on 16 selected peptides showed stable average retention times, further proving the reliability and precision of IonOpticks columns.
Figure 6: Scatter Plot and histogram analyzing the coefficient of variation of retention times for precursors: each dot represents an individual precursor across all of the 257 HeLa QC runs. The plot shows that most data points cluster below a CV of 6%, with the median CV marked at 2.52, indicating consistent retention times across all precursors.
Average retention time for the top 16 peptides over 4 months
Figure 7: Stable peptide average retention times across 4 month period: 16 peptides were selected and their average retention time assessed across 4 months period from HeLa tryptic digest injections on Aurora Rapid 8 cm x 150 μm columns. Samples were run on an Evosep One and Thermo Fisher Orbitrap Astral Mass Spectrometer.
Methods
LC-MS Analysis
A HeLa tryptic digest (200 ng) was separated on Aurora Rapid 8 cm x 150 μm column on a 60SPD method using
Evosep One and Thermo Fisher Orbitrap Astral Mass Spectrometer.
MS parameters: mass range of MS(Orbi)/MS2(Astral) from 380 to 980, isolation windows of 2Th, a maximum
injection time (IT) of 3 ms, FAIMS compensation voltage (CV) set at -40, and a resolution for MS1 set at 120,000.
Data Processing
Data was processed using DIA-NN software (v1.8.1) with match-between-runs enabled. The pg.matrix.tsv, stats.tsv and pr.matrix.tsv tables were used to calculate
protein and peptide identifications and metrics.
Conclusion
The combination of the IonOpticks Aurora Rapid 8×150 column, the Evosep One and Thermo Fisher Orbitrap Astral Mass Spectrometer enabled maximum protein and peptide identifications whilst also ensuring robust and reproducible protein quantification across a large patient plasma cohort analysis.
Acknowledgements
We thank Professor Matthias Mann and his team at the Max Planck Institute of Biochemistry, where all data was acquired and processed. Special thanks to Dr. Igor Paron for his assistance with the LC-MS setup and operation.
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
For further resources and technical support, visit our Help Centre at helpcentre.ionopticks.com. To view other application notes, read the latest publications featuring Aurora Series columns, or view the full range of IonOpticks products, visit our website at www.ionopticks.com
