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Barocycler® NEP2320-45K Extreme

MS Sample preparation by Pressure Cycling

Manufacturer: Pressure BioSciences Inc.

The new bench-top model Barocycler NEP2320-45K EXT from Pressure Biosciences (USA) serves by its Pressure-Cycling process (PCT)* for the standardization and for the accelerated sample preparation in the Proteome Analytics. 

1. Application for protein digestion:


Enzymatic digestion accelerated by PCT*: Trypsin, Lys-C, Chymotrypsin, Glu-C, PNGase F, Lysozyme.

The benefits of the PCT* for sample preparation for the mass spectroscopic analysis are:

  • Huge saving of time sized down from hours to minutes 
  • Better reproducible results at all
  • Optimized and standardized, fast physical-chemical digestion conditions 
  • Lipophilic and membrane connected proteins are better detectable
  • Protein which are difficult to digest are detectable
  • No increase in non-specific cleavage, no oxidation and deamination
  • Repeated runs at the same day are possible

Mass spectroscopic results from thousands of proteins are much faster achievable with PCT*. Results are much more significant and reproducible compared to conventional methods of sample preparation.

2. Application for biopsy tissue:


Protein based measurements in clinical studies and in research need at first a reproducible, efficient extraction of proteins out of small, mostly clinical single samples like needle biopsies, frozen tissue aliquots, microtome cuts, formalin-fixed-paraffin-embedded tissue (FFPE) or just cell suspensions.

The benefits of the PCT* for tissue homogenization and cell lysis are:

  • Very small tissue amounts are sufficient (about 2 mg)
  • Fast and efficient lysis in max. 60 min
  • Physical-chemical process with very reproducible and optimized conditions 
  • Lysis under hypoxic conditions in air tied vials possible
  • Also for FFPE samples optimized
  • Single use vials exclude cross contamination 
  • Follow-up digestion in the same vial
  • Total process time of lysis and digestion: 2 to 4 hours 

 

The cooperation with Prof. Aebersold from the ETH Zürich did lead in the past years to a new developed method based on PCT* which gives an acceleration and standardization in the sample preparation of biopsy tissue before the mass spectrometric analysis. Here a very small piece of tissue is lysed mechanically by PCT-processing: proteins are extracted and afterwards in the same vials the accelerated digestion is executed (see point 1.). By this the sample has a very short preparation time, which takes place under defined, optimized conditions. 

Instrument Engineering:

The Barocycler is equipped with a high pressure cylinder, which is filled with water in which the sample vials are located. During the PCT* process the samples see alternating for some seconds the high pressure – depending on the application between 20 KPSI and 45 KPSI (3100 bar) – and then again normal pressure. The pressure build-up happens in parts of seconds in a patented, mechanical operation and the transfer of pressure is an extremely fast procedure: the pressure is transferred by the process water with the velocity of sound onto the vials called Micro Tubes, which are deformed. The process water can be tempered between 4°C and 95°Cdepending upon the application. The tempering can occur either by a heating mat or by tempered water from a chiller depending on the version of the model. 

The new Barocycler NEP2320-45K EXT is featured by:

  • Temperature range: +4°C up to +95°C
  • Up to 16 Micro Tube samples in parallel processing
  • User programmed pressure profile option: rectangular, sigmoid and others
  • Touch-Screen computer (based on Windows) for handling and data management
  • Display shows real time p/T profiles
  • USB port for data export and Software Updates
  • Network connection optional

We love to provide you more detailed information.

*: The Pressure Cycling Technology (PCT) represents an effect of high pressure on the macroscopic and the molecular level on a water-based, biological sample. The macroscopic effect is mechanical: the compression of the sample vial by the outside pressure crushes the tissue inside the vial. 

The effect on the molecular level is based on high hydrostatic pressure, which accelerates protein digestion via a combination of two mechanisms: The first is the pressure-induced partial denaturation/unfolding and hydration of the target proteins, which leads to better access of the enzyme to its target sites. The second mechanism is less well understood, but relies on the increase in enzyme activity observed for some enzymes at elevated pressure. This second mechanism is likely due, at least in part, to the positive effect of hydrostatic pressure on all reactions that involve hydrolysis. The combination of the two pressure-based mechanisms, one acting on the enzyme and the other acting on the substrates, leads to significantly accelerated digestion.

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