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Application Note: DDA High-throughput proteomics for plasma samples using the Aurora Rapid column



Ultra-sensitive, high-throughput proteomics

IonOpticks’ Aurora Series 5cm Rapid UHPLC packed emitter columns featuring our revolutionary nanoZero® fitting, allow for simplified plug-and-play high-throughput proteomics. These columns are capable of analysing 180 samples per day with a 5 min gradient. Tryptic digested Human Plasma was injected and separated using our 180 samples per day method and data dependent acquisition using PASEF resulting in >230 protein identifications (>1,600 unique peptide identifications) [Fig1, Fig 2] with highly reproducible peptide retention times between runs [Fig 3].

Fig 1 Number of unique peptide IDs

Fig 2 Number of unique protein IDs

Fig 3 Retention time comparison for 6 different peptides across 180 samples

Fig 4 Median and standard deviation of FWHM for peptides

Fig 5 Pearson correlation plot of peptide intensities


Human Plasma tryptic digest was reconstituted in 2% acetonitrile/1% formic acid in MilliQ water to a concentration of 50 ng/μL. Samples were analysed on a M-class (Waters, USA) coupled to a timsTOF Pro (Bruker) equipped with a CaptiveSpray source. 50ng (1μl) of peptides were separated on an Aurora Series 5cm X 150μm Rapid UHPLC packed emitter column, using a constant flow rate of 2μl/min. The column was maintained at room temperature. Sample was injected into a sample loop which takes approximately 0.5min. Mobile phase at 100% buffer A continues to flow over the analytical column during this period facilitating column equilibration. The sample loop was switched on-line for 1min at 100% buffer A. A recommended sample gradient is shown here.

Separately, 20μg of the Human Plasma tryptic digest was resuspended in 10mM Ammonium Formate pH 10. Peptides were separated into 12 fractions using a stage-tip containing 4 X C18 plugs. Fractions were lyophilised to dryness using a CentriVap (Labconco) before reconstitution in 2% ACN, 1% FA prior to analysis. Fractions were analysed using the same LC-MS method as the single shot samples.

Data was analyzed by MaxQuant software using the integrated Andromeda search engine and searched against the human Uniprot Reference Proteome and matched to a high pH fractionation library using the Match-Between-Runs feature.

Aurora Rapid 5×150, 5 minute gradient

(5cm x 150μm ID, 1.6μm C18) Part No. AUR2-50150C18A

Robust reproducibility

• High-throughput analysis

• Maximum protein IDs

• Minimum peak widths

• Consistent performance over large sample cohorts


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Aurora Series UHPLC packed emitter column user guide

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.