The pI-Trap åcelus is the second generation of a sample prefractionation device jointly developed by Biomotif AB and the Karolinska Institute. It provides separation of complex mixtures based on capillary isoelectric focussing (cIEF). This separation process is completely orthogonal to the commonly used hydrophobicity based HPLC separation. The system allows unattended separation overnight and collection of typically 20-25 fractions per sample.
The separation takes place in a unique multi-junction pI cell, which allows the separation even extremely complex mixtures such as serum or plasma. The open tube setup avoids clogging of the system as it can be easily flushed. Samples are separated in an ampholyte free solution, are automatically collected in well plates in a refrigerated environment and can be used for further sample processing such as digestion or direct analysis. The ampholyte free samples are particularly well suited for direct mass spectrometric analysis.
The typical separation time scale is about 1h per sample, shorter turnaround times are possible depending on the actual separation problems. For further subsequent sample processing, the system can handle in excess of 100µg protein in a single separation, which even allows for rapid microLC separations in a clinical proteomics setup for hundreds of samples.
- Sample clean-up and simplification for proteomics
- Clinical biomarker research
- QC of biologics (e.g. screening for Lys presence at mAb heavy chains or pyroglutamate formation)
- Analysis of glycosylation heterogeneities, analysis of sialic acid content and distribution
- Analysis of deamidation processes (converting Asn to Asp or Gln to Glu)
- Significantly higher ID numbers in proteomics experiments, >40% more ID’s from human cell digest in TMT based quantitation experiments on QExactive plus, 70% more protein ID’s on human cell digest in LF quantitation experiment on Orbitrap XL
- Sample in ampholyte free buffer, ready for direct mass spectrometric analysis
- Robust open tube system with minimal losses (>90% recovery rate)
- Much higher sample load than capillary LC allowing for prefractionation with subsequent analysis of the individual fractions with the need for repetitive fraction collection