Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Method development for clean-up of OCT-embedded tissue samples using mixed mode SPE cartridges (#627)

Jennifer M. S. Koh 1 , Natasha Lucas 1 , Dylan Xavier 1 , Andrew Robinson 1 , Steve G. Williams 1 , Peter G. Hains 1 , Phil J. Robinson 1
  1. ProCan, Children's Medical Research Institute, The University of Sydney, Westmead, NSW, Australia

Solid phase extraction (SPE) is important in proteomics workflows to remove impurities and concentrate the peptides before they are analysed by liquid chromatography-mass spectrometry (LC-MS). Optimal cutting temperature (OCT) is a specimen matrix of liquid polymers used for cryostat sectioning of fresh frozen tissues. However, OCT must be removed prior to LC-MS since the polymers compromise the equipment and analyses. OCT is typically removed by tedious washing steps over 1-2 hours, with some degree of sample loss with each wash step. Tissue lysis and digestion follows OCT removal then the digests are passed through reversed-phase C18 cartridges. C18 resin has a strong affinity for OCT (i.e. polymers); hence if any OCT remains after washing, it will outcompete the peptides, leaving a poorly recovered sample which is highly concentrated with OCT and not suitable for LC-MS.

We present a new SPE method using mixed mode strong cation exchange (MCX) cartridges for effective OCT removal. We optimised the method to maximise the removal of OCT and minimise the loss of peptides during SPE. OCT-embedded 30 µm rat kidney sections and human prostate tumour sections were lysed, digested and cleaned up using MCX cartridges. The digests were analysed on Triple TOF 6600 mass spectrometers equipped with micro-LC systems (SCIEX). The MCX cartridges effectively removed OCT from all digests. The presence of up to 85% OCT in the initial tissue samples did not affect lysis or digestion efficiency performed in a Barocycler. The identified proteins and peptides were comparable to those obtained from samples cleaned with C18 (after standard OCT removal).

MCX SPE allows tissue samples to be processed and analysed in a much shorter time frame by completely eliminating manual washing of OCT-embedded sections. The new method is reliable and reproducible, and also minimises the operator’s risk of exposure to biological samples.