Introduction
Advances in mass spectrometry (MS) instrumentation have enabled routine analysis of complex protein samples. However, sample preparation methods are not standardized with many protocols taking 8-24hrs in addition to suffering from low peptide yields, poor digestion efficiency and limited scalability. Recently, we developed a new, simplified sample prep kit containing pre-formulated reagents and a standardized protocol for processing 10µg to 100µg protein samples in less than 2 hours. In this study, we adapted our chemistry to use an automated liquid handling system for sample preparation, TMT labeling, and a 96-well filter plate for peptide clean up.
Methods
Cellular protein extracts and human plasma samples were diluted in lysis buffer. A universal nuclease was added to cellular extracts to reduce sample viscosity. Protein samples were reduced/alkylated before digestion using a trypsin/LysC protease mixture. After labeling with Thermo Scientific™ TMTpro™ 16-plex reagents, a mixed mode peptide clean-up procedure using a 96-well filter plate format was evaluated using a vacuum manifold or automated positive-pressure system. Peptides were quantified and normalized using the PierceTM Quantitative Colorimetric Peptide Assay prior to 1:1 mixing and LC-MS analysis using a Thermo Scientific™ Q Exactive™ HF hybrid quadrupole-Orbitrap™ mass spectrometer.
Preliminary Data
Although our optimized protocol using in our EasyPep kit significantly reduces both hands-on and total sample processing time, peptide clean up using microcentrifuge spin columns is still time consuming with larger sample numbers. We developed a new 96-well filter plate format compatible with TMTpro reagents to support higher sample processing throughput for multiplexed quantitation. Compared to the manual labeling and spin column protocol, this format showed nearly identical performance and quality with scalability and better reproducibility among replicates.
Conclusion
We demonstrate that our chemistry is readily adaptable to automated liquid handling system which provides excellent reproducibility and greatly simplifies proteomic sample preparation.