Freedom EVO® DigestConvenient and robust in-gel digestion and MALDI target spotting

Your two-dimensional gels are running successfully, revealing thousands of spots, each containing an unknown protein to identify by peptide mass fin-gerprinting (PMF). Preparing samples for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS) is tedious work with multiple wash and incubation steps, and is a serious bottleneck for any large-scale proteome analysis project. Can you cope with the number of samples, while avoiding human error and keratin contamination adding to your work with repeated experiments? Can you handle the workload, yet still find time to analyze, write up and publish your results, and plan your next round of experiments?

To add to the challenge, low protein amounts are difficult to detect reliably, or yield low sequence coverage; the result is that many proteins cannot be identified or, even worse, get misidentified. With laboratory resources stretched to the limit, strate-gies to improve overall working efficiency would be desirable by reducing costs, saving time and maxi-mizing use of bench space.

Automated solution Two-dimensional gel electrophoresis is a skilled technique, but it is just the beginning of the work-flow in protein identification by PMF. The whole process, from destaining of gel plugs, reduction, alkylation and in-gel tryptic digest of the proteins, elution of the peptides from the gel, to spotting them onto the MALDI target plates, is tedious and time consuming. Automation of these steps would increase your laboratory’s capacity to pro-cess samples faster, and allow you to perform other tasks, such as analyzing and summarizing your exciting research results.

Based on the Freedom EVO liquid handling work-station, Tecan’s protein digestion workstation is designed for automated processing of both in-gel digestion and MALDI sample preparation, on a sin-gle platform. Tecan also offers established, opti-mized and standardized protocols for the most efficient use of reagents and samples, saving you the costly and time-consuming process of imple-menting the system and developing your pro-cesses. By using standard protein digestion plates, cost of consumables is also kept to a minimum.

The protein digest workstation is capable of over-night runs, processing up to 192 samples in a working day, improving the throughput compared to manual procedures. Scheduling software can increase automated throughput further without changing the technical set-up.

Automated in-gel digestion and MALDI TOF MS sample preparation

2D gel electro - phoresis

Destaingel-plugs

Reduction AlkylationIn-gel- trypsin digest

Spot onto MALDI target

Peptide extraction

MALDI-TOF-MS

96-well protein digestion plate with 250 μm holes (AB-gene® (Thermo Fisher Scientific))

One platform for digestion and spottingThe versatile Freedom EVO platform is able to per-form the whole process from gel destaining to matrix spotting, by automation, saving costs and space in the laboratory.

Reduction in keratin contaminationThe minimized hands-on time reduces the risk of keratin contamination, improving reliability of results. Contact-free pipetting combined with the continuous protection of the silicone mat cover also helps to minimize contamination. The optional dust cover allows full enclosure of the robotic workstation, further reducing contamina-tion risk.

Unique in-gel digestion stackA specially designed in-gel digestion stack is made up of four components, including a 96-well protein digestion plate developed for this purpose by AB-gene® (Thermo Fisher Scientific). Each well of the di-gestion plate has two 250 μm holes to allow removal of reagents using the Te-VacS™ vacuum separation module. The protein digestion plate fits into a holder on a frame and is covered with a metal plate perfo-rated with 96 holes. A pre- perforated silicone mat on the underside of the metal plate protects the sam-ples from evaporation and contamination and can be penetrated by the pipetting needles, while the in-trinsic weight of the metal lid prevents the stack from lifting during withdrawal of the pipetting needles.

Contact-free pipetting through the silicone mat cover. The silicone mat prevents evapo-ration and can be removed to allow evaporation during drying steps.

Protein digestion on Freedom EVO. The worktable includes heated and cooled devices, as well as a Te-VacS vacuum separation module.

In-gel digestion stack components: lid including silicone mat, standard digestion plate, plate holder and base frame

Decreased hands-on timeAutomation achieves considerable labor savings and eliminates human error. Both the automated and manual in-gel digest processes need approxi-mately one hour of preparation time but, in con-trast to the automated procedure, the manual method requires further hands-on intervention, tying up one person almost full-time for about seven hours.

Robust and repeatable resultsIn-gel digestions and sample preparations of BSA at three different concentrations have been per-formed, using both manual and automated meth-ods. The results are shown on the figure to the right. Bars represent means of two independent experiments run with twin samples (n = 4), and the error bars represent standard deviation. The results demonstrate that the platform is capable of equal or better PMF sequence coverage, compared to the manual procedure, achieving excellent sensitivity of down to 50-100 fmol/gel plug.

MALDI target spotting using fixed tips or Zip Tips®MALDI target spotting is possible using Zip Tips® (Millipore) for desalting. Alternatively, by using fixed tips for layer affinity preparation, the costs for consumables can be reduced significantly. Wash procedures preventing carryover of peptides are available.

Time required for in-gel digestion of 192 samples by automated (A) and manual (M) process. Preparation time is shown in green, and subsequent hands-on times in red.

The Freedom EVO Protein Digest Workstations support MALDI targets of all relevant manufacturers.

Sequence coverage of PMF of BSA at three concentrations, comparing in-gel digestion by automated (yellow) and manual (white) methods.

0 100

A

M

200 300

time (min)

400 500

Sequ

ence

cove

rage

(%)

Protein conc. (fmol/gel plug)

BSA

5242 524 520

20

40

60

80

Gel plug handling with minimal risk of cross-contaminationContactless pipetting and removal of reagents prevent impaling the gel plugs with the pipetting needles, and thus the accidental loss of samples. Furthermore, it eliminates carryover from one well to another.

For extraction, tryptic peptides are collected by passive elution and recovered on the Te-VacS vacuum separation module into a 96-well collect-ing plate, allowing for maximized recovery of the peptides1.

Unique cooling/heatingThe specially designed in-gel digestion stack may be assembled using all four components or without the lid and/or base frame. This not only allows for cooling after runs, and therefore make

overnight runs possible, but also to either allow or prevent evaporation during incubation steps, de-pending on the process step. Special cooling and heating devices for these process steps are available.

Digestion stack (green) on the vacuum module (yellow) for pipetting and aspiration of reagents.

Digestion stack on the vacuum mod-ule for the elution of tryptic peptides into the collecting plate

Worktable of a Protein Digest Workstation including heating and cooling devices.

Freedom EVO specifications

Recommended optional parts

General hardware featuresRobotic arms, Liquid handling arms, robotic manipulators, several multi-channel optionsTip configuration 1, 2, 4, 8 tips, various combinations of application-oriented tip typesTip types Standard (Teflon®-coated stainless steel) and disposable tips with or without filter (10/200/1000 μl); low-volume tips for high-density format applicationsSyringe sizes 25/50/250/500/1000/2500/5000 μlLiquid handling featuresVolume range 0.5 μl – 5000 μlPipetting precision Volume Standard tips Disposable tips lalal 200 μl 1000 μl 10 μl ≤ 3.0% ≤ 3.0% – 100 μl ≤ 0.5% ≤ 0.5% ≤ 1.0%Safety Features Liquid detection Presence, absence and quantification check of liquid volumes is available for all tip types, various detection modes (single/multi) selectableClot detection Detection of tip-obstruction by solid materialPassword protection Three password levels: Administrator, Application Specialist, Operator

Reference1 Olaf Jahn, Dörte Hesse, Marina Reinelt, Hartmut D. Kratzin: Technical innovations for the automated identification of gel-separated proteins by MALDI-TOF mass spectrometry. Anal. Bioanal. Chem. (2006) 386: 92-103

Tecan Group Ltd. makes every effort to include accurate and up-to-date information within this publication, however, it is possible that omissions or errors might have occurred. Tecan Group Ltd. cannot, therefore, make any representations or warranties, expressed or implied, as to the accuracy or completeness of the information provided in this publication. Changes in this publication can be made at any time without notice. All mentioned trademarks are protected by law. For technical details and detailed procedures of the specifications provided in this document please contact your Tecan representative. This brochure may contain reference to applications and products which are not available in all markets. Please check with your local sales representative. Scientific instrumentation. Not for use in human or clinical applications.

© 2008, Tecan Trading AG, Switzerland, all rights reserved.Tecan is in major countries a registered trademark of Tecan Group Ltd., Männedorf, Switzerland

Freedom EVO, Te-VacS are in major countries trademarks or registered trademarks of Tecan Group Ltd., Männedorf, SwitzerlandZip Tip is a registered trademark of Millipore. ABgene is a registered trademark of Thermo Fisher Scientific. Teflon is a registered trademark of E.I. DuPont.

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The optional dust cover is available for Tecan Freedom EVO robotic workstations in the sizes of 75, 100, 150 and 200 cm. Two passive ventilators containing a filter allow circulation of air and heat. The dust cover seals against the safety panels of the robotic workstation to create a full enclosure, preventing air-borne particles from falling onto the worktable. The risk of keratin contami-nation of your valuable samples is therefore reduced to a minimum, making the dust cover a perfect tool for ensuring robust and reliable results.

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