High-throughput proteomic sample preparation using pressure cycling technology

Abstract

High-throughput lysis and proteolytic digestion of biopsy-level tissue specimens is a major bottleneck for clinical proteomics. Here we describe a detailed protocol of pressure cycling technology (PCT)-assisted sample preparation for proteomic analysis of biopsy tissues. A piece of fresh frozen or formalin-fixed paraffin-embedded tissue weighing ~0.1–2 mg is placed in a 150 μL pressure-resistant tube called a PCT-MicroTube with proper lysis buffer. After closing with a PCT-MicroPestle, a batch of 16 PCT-MicroTubes are placed in a Barocycler, which imposes oscillating pressure to the samples from one atmosphere to up to ~3,000 times atmospheric pressure. The pressure cycling schemes are optimized for tissue lysis and protein digestion, and can be programmed in the Barocycler to allow reproducible, robust and efficient protein extraction and proteolysis digestion for mass spectrometry-based proteomics. This method allows effective preparation of not only fresh frozen and formalin-fixed paraffin-embedded tissue, but also cells, feces and tear strips. It takes ~3 h to process 16 samples in one batch. The resulting peptides can be analyzed by various mass spectrometry-based proteomics methods. We demonstrate the applications of this protocol with mouse kidney tissue and eight types of human tumors.

ASAP─Automated Sonication-Free Acid-Assisted Proteomes─from Cells and FFPE Tissues

“…They can be archived for long-term storage at room temperature without compromising tissue integrity and architecture. Several studies have successfully implemented mass spectrometry-based shotgun proteomics on FFPE specimens, leading to a significant advancement toward clinical proteomics. ,, However, these studies depend on high amounts of the FFPE tissue starting material, expensive equipment, and peptide labeling reagents for sample processing that are not usually accessible in clinical settings, which requires fast sample analysis for target identifications. Sample processing of FFPE specimens also involves tedious workflows involving multiple processing steps to remove the LC-MS incompatible reagents from the lysates, resulting in inevitable sample loss, and is time-consuming, making it challenging to be suitable for high-throughput studies.…”

Identifying patients with rapid progression from hormone-sensitive to castration-resistant prostate cancer: a retrospective study

“…The PCT-assisted proteomics sample preparation procedures followed our previously published workflows 13 . In brief, about 0.2 mg of FFPE punches were dewaxed with heptane, hydrated with ethanol, and then underwent acidic hydrolysis by 0.1% formic acid (FA, Thermo Fisher Scientific, USA) and basic hydrolysis by 0.1 M Tris-HCl (pH = 10.0).…”

Proteomic profiling of ovarian clear cell carcinomas identifies prognostic biomarkers for chemotherapy

“…To remove the optimal cutting temperature (OCT) compound in FF samples from H2 cohort, we first washed each sample (∼1 mg) in sequential concentrations (volume/volume) of ethanol-water as following: 70% (30 s), 0% (30 s), 70% (5 min, twice), 85% (5 min, twice), and 100% (5 min, twice). Then, the FFPE and FF samples were subjected to PCT-assisted lysis and digestion using protocol following workflow B described by Cai et al [11].The resulting peptides were desalted using C18 columns (The Nest Group, Inc., MA) according to the instruction of the manufacturer and reconstituted with 2% acetonitrile (ACN) and 0.1% formic acid (FA).…”

“…We performed dewaxing and de-crosslinking for the FFPE samples as described previously [11,12,24]. To remove the optimal cutting temperature (OCT) compound in FF samples from H2 cohort, we first washed each sample (∼1 mg) in sequential concentrations (volume/volume) of ethanol-water as following: 70% (30 s), 0% (30 s), 70% (5 min, twice), 85% (5 min, twice), and 100% (5 min, twice).…”