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My MP involves the analysis of Stenotrophomonas maltophilia’s secretory and cell surface proteins. This would involve the use of quite a number of machines. The one that I am going to blog about is Xcise. We were given a training that lasted for 3 days. The first day is a trial run, the second day is about how to use the software and the last day is more on the maintenance of the machine.Xcise is an automated gel processor that is able to process proteins that are to be identified by mass spectrometry, from the acquisition of gel image to the spotting of protein sample onto a MALDI target plate.
This picture is taken from http://www.ssi.shimadzu.com/products/images/Biotech/xcise.jpg
Terms:
2D gel electrophoresis: Proteins are separated twice. For the 1st dimension, proteins are separated using an IPG (Immobilized pH Gradient) strip based on their isoelectric point. The proteins move horizontally. For the 2nd dimension, proteins are separated using a pre-casted gel based on their molecular weight. The 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.
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 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 to be visualized and 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
- Gel image is acquired and spots to be cut are selected
- A cutting head will cut out the gel containing the protein spots and place them into wells
- The gel will then be destained and dehydrated
- Trypsin is added to digest the proteins into peptides (note that the proteins are still within the gel)
- Sample is then incubated for 4-6 hrs at 37oC or 16-18 hrs at 30oC
- As buffer used contain salts that will affect the subsequent analysis of peptides in MALDI, peptides would need to be desalted
- 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
- The peptides are then spotted onto a MALDI plate together with a matrix compound
- The MALDI plate can then be placed in MALDI and peptides can be analysed
LMQA – Lab Automation
Using of Xcise is an example of lab automation. If the process is to be done manually, it will be very tedious. For the cutting of gel, the lab technician would need to measure the diameter of the protein spot, cut a pipette tip so that the hole corresponds to the diameter and use the pipette tip to cut the gel. The gel would be inside the tip and would have to be taken out using a pointed end. Steps 3-7 would need to be done manually too by adding and removing the required solutions. 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.
However, if Xcise is used, the cutting of gel would be faster and more precise. At the spotting stage, Xcise can spot 8 samples at a time, which is much faster and accurate than doing it manually. Contamination is also greatly minimized. So 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.
This picture is taken from http://www.millipore.com/micro/jp/zt/ziptip.gif
That is all about it. Feel free to ask any questions. Thanks=]
Chua Ming Boon
Tg01
0503197F
14 comments:
Halo!
I'm kinda lost. Step 7 onward rite, u mean the ziptip is already removed from Xcise?then, the spotting of the maldi plate is done in Xcise itself?or spotting had to be done manually before loading of the plate into the machine?
charmaine yeo
TG01
Charmaine
No, ZipTip is only used in step 6 to suck up the sample for desalting. In step 8 after the spotting then the ZipTip will be removed. If Xcise is used, spotting of MALDI plate will be done by Xcise itself. However, if you do it manually (not using Xcise), you will have to use a micropipette to spot the matrix onto the plate first, and then the sample is spotted ontop of the matrix.
Regards,
Ming Boon
hihi! wah so fast u started your mp, i'm still thinking of the title!
yah have to agree with charmaine is kinda confusing. ok the qns...
1)step 3 how and why u destain the gel? issit got to do with result interference?
2)step 5 which incubation is recommended? any pros and cons?
good to see that everyone of us have a fruitful experience. good luck for your mp! do visit my blog to check for updates =)
Chaur Lee
TG01
Yup. Because our's is research de..
Destaining is done by using ammonium bicarbonate(NH4HCO3). The stain is -vely charged and will bind to the proteins that are +vely charged. NH4HCO3 will make the environment alkaline and the proteins will become neutral. Thus the stain(-ve) would not bind to the proteins anymore. Stain is removed because it will affect the anaysis of peptides in MALDI.
It depends on the situation, no particular pros and cons. Lets say if by the time i need to add the trypsin, its already 5pm. Then i would choose the 30 degrees celsius for 16-18 hours so that i can come back tml morning at about 9am to stop the digestion. If i use the 37 degrees celsius one, i would need to wait until about 9pm to stop the digeston, which is not very practical.
Good luck for your MP too and hope that your title can be comfirmed soon!
Regards,
Ming Boon
hey ming boon,
what are the maintenance steps taken for the Xcise machine?
-sharon ang.tg02
hi mingboon,
your mp sound fun.
i would like to ask is there any disadvantage or limitation for the machine, Xcise.
Lizzie
Sharon,
The maintenance steps are quite simple. Just need to ensure that the 2 bottles of MilliQ water are filled. One bottle of MilliQ water is for the cutting of gel and another bottle is for flushing of probes. When the machine is started up, must always run auto-home routine, where the machine will prime the tubings and self-calibrate. After using the machine, the plastic frame to place the gel must be washed with 70% ethanol. Cannot wash with acid because the acid will corrode the frame. That should be about it.
Hope you are doing well=] Take care~
Ming Boon
Lizzie,
Basically, the machine is quite good except for one disadvantage. After cutting the gel, the cutting head will put the gel in the well. But when the cutting head pushes the gel into the well, if the force is too big, there is a possibility that the gel will bounce out from the well. The lab tech would have to sit there and check that it does not happen so its not that automated.
Ming Boon
Heyy!
Your MP title sounds really cool. Hm. Seems like enough questions has been asked about the machine.As for the 2D separation, I was wondering how you actually go about doing the 1st dimension of the 2D separation, using the IPG strip.
What are the advantages and disadvantages of using IPG? I heard that some larger proteins may also be lost during this process? Please enlighten, thanks!
Kent TG01
IPG strip is a thin strip of gel casted on a plastic backing. Proteins enter the gel through rehydration. There are 3 types of method to do it. Passive rehydration, active rehydration and cup loading. We will be using the passive rehydration, where the gel is placed in contact with the protein sample for at least 16 hrs and the proteins will be absorbed into the gel passively. The rehydration will be done in a tray. After the sample is loaded, the strip will be transferred to a focusing tray that is to be placed in PROTEAN IEF cell, where a voltage will be applied.
Advantages:
-Convenient because bought commercially, last time the gel has to be prepared by the user.
-The gel is supported by the plastic backing while last time, the gel is very fragile.
-Strip can be stored prior to use while last time, gel has to be casted prior to experiment.
-Higher amount of proteins can be loaded as compared to last time.
-Has a higher throughput as up to 12 11cm/17cm strips or 24 7cm strips can be run at one time, while last time, one gel can only run one sample.
Couldn't think of any disadvantages.
Agarose gel is preferred as it has a larger pore size. In the case of IPG strip, the gel used is polyacrylamide(I don't know why), which has a smaller pore size, preventing the larger proteins to enter the gel. But this can be minimised by choosing a lower gel concentration(larger pore size).
Hope this answers your questions=]
Regards,
Ming Boon
Kent!!
I found out the disadvantage le. The main problem is that hydrophobic and basic proteins will be lost because they cannot enter the gel.
Ming Boon
hi!!
you mentioned that sample is then incubated for 4-6 hrs at 37oC or 16-18 hrs at 30oC. The incubation is crucial for wat process?? the purpose to incubatE?
And also how are the peptides desalted??
Vinodhini
TGO1
Hi,
Erm, i mentioned in the previous step that trypsin is added right. The incubation is to allow the trypsin to digest the proteins into peptides. The peptides will then be analysed by MALDI (Proteins would be too complex to be analysed so must digest them into peptides).
Peptides will be desalted using a solution, 0.1% Trifluoroacetic acid (TFA). I am not sure of how the TFA desalts the peptides. Will update when i find out the answer.
Ming Boon
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