Introduction to the Topic: My MP involves the analysis of Stenotrophomonas maltophilia’s periplasmic proteins. Periplasmic proteins are proteins that are found between the the inner and outer cell wall. The study of these proteins are important as it could be potential drug targets in the future. During the course of this study, quite a number of machines would be needed.
The machine im going to blog about in this entry is the Xcise machine
So what is Xcise? Xcise is an automated gel processor that is useful in processing proteins that are to be identified by mass spectrometry. Its functions ranges from acquisition of gel image to the spotting of protein sample onto a MALDI target plate. Before we can use Xcise, 2-D gel electrophoresis should be performed 1st.
2D gel electrophoresis: Proteins are separated twice during gel electrophoresis. For the 1st dimension, proteins are separated using an IPG (Immobilized pH Gradient) strip according to their isoelectric point. The proteins move horizontally. For the 2nd dimension, proteins are separated using a pre-casted gel based on their molecular weight. This time, proteins move vertically downwards.
Isoelectric point: Isoelectric point is a characteristic of the protein whereby it corresponds to the pH at which the protein is neutrally charged. This means that the proteins will migrate on the IPG until it reaches the pH of the strip, whereby the protein is neutrally charged.
Mass Spectrometry: A technique used to identify and sequence proteins by measuring the mass-to-charge ratio of proteins that are converted to ions. The instrument used to measure the mass spectrum is called MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight).
Matrix compound: A compound that is required to control the energetics of the desorption/ionization process.
After a gel is run, proteins are already separated based on their isoelectric point and molecular weight (2D gel). Proteins separated would appear as spots on the gel. The protein spots would then be stained for visualization and a gel image is acquired. After the gel is stained, we would want to identify the proteins. Before running the proteins through MALDI, the proteins would need to be removed from the gel, digested into peptides and then spotted onto a MALDI target plate.
Outline of Xcise’s in-gel digestion procedure
1. Gel image is acquired and spots to be cut are selected
2. A cutting head will cut out the gel containing the protein spots and place them into wells
3. The gel will then be destained and dehydrated
4. Trypsin is added to digest the proteins into peptides (note that the proteins are still within the gel)
5. Sample is then incubated for 4-6 hrs at 37oC or 16-18 hrs at 30oC
6. As buffer used contain salts that will affect the subsequent analysis of peptides in MALDI, peptides would need to be desalted
7. Peptides are then eluted using ZipTip. ZipTip is different from a normal pipette tip as it contains a resin at the tip. Peptides would bind to the resin during the process of desalting
8. The peptides are then spotted onto a MALDI plate together with a matrix compound
9. The MALDI plate can then be placed in MALDI and peptides can be analysed
LMQA (How can excise be helpful?)
Using Xcise is an example of lab automation. If the process is to be done manually, it will be very tedious and time consuming. For the cutting of gel, the lab technician would need to measure diameter of the protein spot, before cutting a pipette tip so that the hole corresponds to the diameter. The pipette tip will then be used to cut the gel. During cutting, the gel would be lodged inside the tip and would have to be taken out using a pointed end. This makes cutting extremely difficult. Steps 3-7 would need to be done manually too by adding and removing the required solutions manually.
For the spotting onto MALDI plate, lab technician would have to spot onto the plate one by one. One spot will be placed in one eppendorf tube so if there are 100 spots, there will be 100 tubes to be processed and 100 spots to be spotted onto the plate. This may lead to fatigue and air bubbles in the sample may be produced. However, if Xcise is used, the cutting of gel would be faster and more precise, hence decreasing the amount of varaitions. At the spotting stage, Xcise can spot 8 samples at a time, which is much faster and accurate than doing it manually. Contamination of the sample is also greatly minimized. Even though the machine and the consumables are very expensive (1 ZipTip costs about $2, and 8 ZipTips will be used at a time), it can greatly improve the efficiency of the lab due to its importance in maintaining the integrity of the sample.
Posted by: Ma Xianwei Benjamin