Today using targeted quantitative proteomics techniques such as MRM analysis or SWATH^® Acquisition, protein panels can be quantified across a broad dynamic range with very high reproducibility. While these techniques have increased the reproducibility and scale of protein analysis, sample preparation remains a key bottleneck as the number of samples increases. Numerous processing steps are required, each susceptible to technical variation. Here, we have used automation to improve the throughput and day-to-day reproducibility of the protein digestion portion of the sample preparation workflow.

To increase throughput and reduce technical variation, we have implemented an automated peptide preparation protocol on a liquid handling workstation (Biomek NX^P) coupled with an MRM workflow using a QTRAP^® 6500 system. Denaturation, reduction/alkylation and digestion were the steps included, total workflow time was ~5-6 hours, depending on # of samples processed. A large number of proteins/peptides were monitored to ensure that good general digestion was occurring. Two different automation workstations were used to confirm the method transferability and multi-day experiments were performed on each to confirm method reproducibility.

First the protocol was validated by performing a very careful manual digestion across 24 wells using MRMs to 150 peptides. Using this benchmark, the automation protocol was optimized to achieve similar or better performance. For off-deck digestion using separate incubators, on average, ~80% of peptides monitored had raw peak area CVs < 10% as monitored by LC/MRM analysis.  Next steps will be to optimize the use of on-deck heating (incubators integrated on workstation) and assess variance. Use of internal standards could also be incorporated for specific workflows to further reduce variance and will be assessed.

The automation method provided excellent reproducibility for digestion across multiple days on multiple workstations.