Scalable and comprehensive protein analysis sample preparation workflows: analyzing fresh frozen tissue samples made easy

Human tissues are an important link between organ-specific spatial molecular information and understanding pathology, which in turn helps in optimizing treatment options. Fresh frozen (FF) tissue is often the preferred sample for proteomics because it preserves proteins in their natural state. However, patient tissues are uniquely obtained by time and location, and limited in their availability and size. Currently, little knowledge exists about appropriate and simplified protocols for routine Liquid Chromatography (LC) – Mass Spectrometry (MS)-based analysis of various types and sizes of tissues.

In this report, we demonstrate the use of a comprehensive sample preparation workflow resulting in generation of high-quality proteins and peptides from FF tissues by leveraging class leading Adaptive Focused Acoustics® (AFA®) Technology. The AFA enabled workflows also employ dedicated consumables that ensure complete homogenization of tissues to extraction of proteins followed by purification and expedited digestion to peptides. The protein analysis workflow detailed in this study highlights use of a novel consumable with R230 focused-ultrasonicator that enhances homogenization efficiency of FF tissues with AFA.

For sample preparation, FF tissue samples (2 mg, 5 mg, and 10 mg) were treated with AFA after they were loaded in the tube (microTUBE-130 with Bead Snap-Cap) with 120 mL of buffer (two different buffers were used - 8M Urea and Tissue Lysis Buffer). In addition to liver samples, small intestine and lung tissue samples were tested using the same workflow. Post-homogenization, the samples were centrifuged using the prototype rack followed by collection of the supernatant. BCA assay was performed with the supernatant to determine total protein concentration. The downstream protein cleanup was performed with either S-Trap or Protein Aggregation Capture (PAC) methods followed by AFA-enabled expedited digestion for generation of peptides. The homogenization tests were performed at 10 °C and in continuous mode with 5 mins of AFA treatment per column.

Different time intervals were used with the above-mentioned AFA settings to ensure complete homogenization of FF liver tissue samples. While 5 mins of AFA treatment was sufficient for complete homogenization of 2 mg and 5 mg samples, 15 mins was necessary for 10 mg of samples. Average protein yield (mg) for every mg of FF tissue samples were 142 (2 mg tissue), 133 (5 mg tissue), and 102 (10 mg tissue) with the %CV not exceeding 9.2. Similar results were observed for Lung (13 mg, 15 mins) and small intestine (11 mg, 15 mins).

Results in this study demonstrates an efficient, reliable, scalable workflow for a wide array of FF tissues focused on complete homogenization of samples and maximizing protein yield, while ensuring high reproducibility. The specific tubes used in this study can be used in a 96-plate format with the appropriate rack that is compatible with a host of focused ultrasonicators