Automation of tissue homogenization for liquid chromatography-mass spectrometry (LC-MS) analysis using the Omni LH 96 automated workstation.

Quantifying protein expression via liquid chromatography-mass spectrometry (LC-MS) has become more widely used in all areas of biomedical research and development. Quantifying protein biomarkers can provide essential information on drug efficacy, mechanism of action, target engagement, and safety [1]. In recent years, LC-MS has been applied in a wide array of research areas, including mRNA, lipid nanoparticles (LNPs) [2], gene therapy (GTx) [3], and protein degraders (PROTAC) [4]. 

LC-MS analysis, when combined with immunoaffinity capture and the use of stable isotope-labeled internal standards, allows for the quantification of protein biomarkers with high sensitivity and unparalleled specificity in complex matrices, including serum, plasma, and tissues. Furthermore, the use of chromatographic separation allows for the quantification of multiple proteins from a single sample. The demand continues to rise to develop and deploy these LC-MS assays quicker than ever, without compromising the consistency and reproducibility of the assay. This has led to a need for automation, particularly in tissue dissection and homogenization, which has been identified as the greatest source of variability during sample preparation for LC-MS [5]. The traditional method for tissue sample preparation (shown in Figure 1) is tedious, time consuming, and labor intensive. It introduces operator variability, making tissue dissection and homogenization an excellent candidate for automation. 

This application note focuses on the benchmarking and implementation of the Omni LH 96 workstation to automate the weighing and homogenization of tissue samples for downstream LC-MS analysis. A series of tests will be outlined using various tissue types to compare the LH 96 automated workstation vs traditional bead milling. Results showed that the LH 96 was comparable or even more efficient than traditional bead milling in homogenizing various tissue types. LC-MS analysis further proved that the two methods are equally efficient and can be reliably applied in a high-quality assay. Implementation of the Omni LH 96 automated workstation resulted in ~40% increase in throughput. The direct workload for analysts was reduced even greater, as there was no longer a need for tissue sectioning or pulverization, and all weighing, LB addition, and homogenization steps were automated. This frees up hours of analyst time to perform other tasks while the LH 96 workstation is running.

For bead mill homogenization, tissues were transferred to 1.5 mL RINO tubes (Next Advance, Cat # TUBE1R5-S), and a small scoop (about 250 mg) of 0.5 mm diameter stainless steel beads (Next Advance, Cat # SSB05) were added to each sample. Lysis buffer (LB) (5% SDS (Fisher Scientific, Cat # BP2436) in 1x RIPA (Millipore Sigma, Cat # 20-188) with protease inhibitors (Cell Signaling, Cat # 5872) was manually added to yield a concentration of 50 mg of tissue (weight) per mL of LB. Samples were processed in the Omni Bead Ruptor Elite bead mill homogenizer (Revvity, Cat # 19-042E) using a standard method. After centrifugation, the supernatant containing protein was collected and transferred to a clean plate. Samples were used immediately or stored at –80 °C until ready to proceed with downstream analysis.

Figure 4: Comparison of tissue homogenization efficiency in different pulverized rat tissues.