Robust sample preparation workflows for protein analysis: removing complexities and variability while ensuring high-quality data

Robust, efficient, and reproducible protein extraction and sample processing is a key step for bottom-up proteomics. While Liquid Chromatography (LC) coupled to Mass Spectrometry (MS) based workflows are sophisticated multistep processes, the resultant data quality essentially depends on the characteristics and limitations of each step employed, from sample preparation to LC-MS analysis. The sample preparation workflow is a key determinant in defining what proportion of the protein/proteome is available for end detection and analysis. Moreover, the robustness of sample preparation workflows control variation and systematic bias.

In this report, we demonstrate the use of a comprehensive sample preparation workflow for generation of high-quality proteins and peptides from mammalian cells and Formalin Fixed Paraffin Embedded (FFPE) tissue samples by leveraging class-leading Adaptive Focused Acoustics® (AFA®) Technology. The AFA-enabled workflows also employ truPREP® Protein kits for cells and FFPE samples, respectively.

Cells: A wide range of mammalian cells (0.1-0.5 M) were harvested followed by aliquoting 25 mL of washed and prepared cell pellets in each well of the 8 AFA-TUBE TPX strips. 25 mL of lysis buffer was added to each sample followed by AFA and centrifugation. Reduction and alkylation of the extracted proteins were performed by addition of reducing agent to each sample followed by heating at 50 °C (1 hr) and addition of alkylation reagent. Purification of samples was done using an optimal amount of magnetic bead mixture. The beads were then suspended in Trypsin/Elution buffer for accelerated digestion. Both purification and digestion processes were enabled by AFA.

FFPE: 10 µm FFPE scrolls were deparaffinized with 50 µL of rehydration buffer and 450 µL Deparaffinization Solution in a prepTUBE TPX, following incubation at 56 °C for 5 minutes. After centrifugation, Deparaffinization Solution was discarded and 100 µL lysis buffer was added followed by homogenization of tissues with AFA in a Covaris R230 Focused-Ultrasonicator. De-crosslinking of proteins was conducted at 90 °C/ 90 min prior to another AFA treatment, followed by reduction and alkylation. Protein purification was conducted following the Protein Aggregation Capture (PAC) protocol with magnetic beads. The beads were suspended in trypsin buffer for overnight digestion. The purification process was enabled by AFA.

The results shown in this study exhibit robust and reliable workflows using kits that can address a wide array of mammalian cells and FFPE samples resulting in high-quality proteins and peptides. The FFPE workflow and kit use a deparaffinization step that does not use any toxic and harsh, protein damaging chemicals, and it generates consistent yields of proteins from only one 10 mm FFPE scroll. For cells and FFPE samples, both protein and peptide yields were found to be very reproducible, with low CVs for intact proteins (~7%) and peptides (~9%).