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1. Tissue/cell disruption and homogenization with Covaris instruments:

In biology, proteomic sample preparation is very difficult to control. The first step of proteomic sample preparation is to thermally stabilize the sample prior to, and during, the deconstruction of the initial sample, usually a piece of tissue in the centimeter scale, to finer particles in the nano- to micro-meter scale, so target molecules, such as nucleic acids, peptides and metabolites can be released from the tissue for further processing. Techniques often compromise the thermal stabilization to achieve "efficient" disruption. For example, bead-beating systems may effectively homogenize, but the grinding mechanism overheats the sample.

In addition, most current mechanical-based sample preparation processes cannot complete the task of deconstruction effectively; usually breaking down the tissue to about 100 micron size range, much larger than the desired single molecule size level. For example, using a mortar and pestle with liquid nitrogen is a good thermal control, but the resultant "powder" is ~100 µm. Thus, most target molecules, such as DNA, RNA or protein,  still trapped in the tissue mass, cannot be extracted out efficiently. These target molecules are dependent on diffusion-limited digestion or dissolution. This is true even for laser captured thin tissue sections. The diverse, multi-faceted aspects of proteomic sample preparation (such as small to large molecular weight, hydrophobic to hydrophilic domains) benefits from a non-contact, isothermal, mechanical-based technology. Based on the target molecule, the extraction buffer may be varied. The Covaris AFA process was designed for challenging and problematic sample preparation.

The Covaris CryoPrep™ system was developed to further enhance the capabilities of the Covaris acoustic systems, such as processing hard tissues such as bones or cartilage. It is a patented microprocessor controlled hammer and anvil mechanism that delivers controlled, repeatable mechanical impact (400 millisecond) to a sample that may be at -80°C. This controlled impact to frozen sample results in true freeze-fracture pulverization, efficiently shatters tissue pieces to a molecular size level, disrupts extracellular matrix, and dramatically increases the samples surface area, which is crucial for further downstream processing. In some cases, this chemical-based process is still used with Covaris instruments. Usually, the Covaris process enables the chemical to reach more sample surface area and make the chemical treatment more efficient.

Sometimes, samples from CryoPrep will undergo further processing with Covaris AFA™ instruments, such as Covaris S-series or E-series, to complete the protein extraction step.

2. Protein Tryptic Digest:

Protein spots from 2D SDS gel usually will undergo Tryptic enzyme digestion before being loaded to Mass Spectrometry. Since this enzyme limit digestion step normally takes over 10 hours, researchers usually perform this tryptic digestion procedure overnight. This is a diffusion limited process, and as such, will be limited to the rate of enzyme substrate replenishment. Given the sample-to-sample variations, this is a difficult process to standardize and control.

With Covaris L-series or E-series, the digestion can be completed in less than 30 minutes, with higher reproducibility, better recovery, and more sample complexity. Using these Covaris systems, standardizes the energy delivered to each sample in an isothermal manner, and standardizes the sample preparation processes.

The graph above shows how "gentle mixing," while using Covaris AFA, can dramatically speed up trypsin digestion—from 16 hour (overnight) digestion to less than one hour:

• 200 µl trypsin digest of plasma protein (0.35 µg/µl) in HEPES, CaCl, pH 8.0, Urea
• 96 well p-propylene microtiter plate