Saturday, July 5, 2008

Week 2 SIP - Xcise Machine

Name of topic: Xcise

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

Class: TG01

10.15am

05/July 2008

12 comments:

De Incredibles said...

Hi Benjamin

I have a qustion for you. U mention that the study of the periplasmic proteins in S. Maltophilia is important as they could be potential drug targets.....however u did not mention why there is a need to study about S. Maltophilia . Thus, my qustion is

What is the threats posed by S. Maltophilia ?

Xin Ni
TGo2
Group 9

tg01 group 2 said...

Hi Xinni

Actually very little is known about this microorganism and this include its pathogenicity and virulence factors. This is because researchers have not found out these mechanisms yet. They only know that it sectrete proteins at 37 and 28 degree celsius that may confer pathogenicty.

S.maltophilia can cause pneumonia, urinary tract infection and septicemia in immunocompromised people.

Thanks for the question and i hope i have answered your question.

The threat of S.maltophilia is that it can cause nosocomial infection (hospital acquired). Thus in a normal healthy person, it will not cause disease. However in a hospital enironment whereby there are lots of immunocompromised people, the use of non-sterilised surgical equipment such as catherers infected with this bacteria can cause disease.

MadTechs said...
This comment has been removed by the author.
MadTechs said...
This comment has been removed by the author.
MadTechs said...

Yoz Ben

Woah, you posted a whole chunk of information on the blog, you must had a tough week there.

Okay, here is my question:
1) Regarding the isoelectric point, does the characteristic of the protein pH cause it to migrate to the part of the IPG of correspond pH and stop there?

2) You mentioned that the protein is neutrally charged, is it important for the IPG process?

3) What is the mode of action of trypsin in digesting the proteins?

4) You mentioned that the gel would be stained for protein identification, what staining solution do you use?

Hahaz! I hope it isn't a lot of question for you to answer =P

Best of luck for your in-house SIP! Have a great time there =)

Quan Jun
TG02
Posted: 07 July 2008

tg01 group 2 said...

Hi Quan Jun,

1) The purpose of using isolelectric point is to determine whether the protein is positively or negatively charge. The misconception people have is that protein are negatively charged.On the IPG strip there is pH gradient of 3 to 10. So if the protein is negative, it will move towards the positive charge until it reaches the pH on the stirp whereby it is neutrally charge and stops there.

2)Yes, when the protein is neutrally charge, it means that is its isoelectric point. And by the formula, pH (meaning the pH to where it moves) is smaller than the pI, the protein is positively charged and likewise vice versa. It is important to determine the pI that correspond to the pH in order to characterise the protein

3)Trypsin cleaves peptide chains at the carboxyl side of the amino acids lysine and arginine, except when either is followed by proline. Typsin is used in proteomics study to digest proteins to peptides for mass spectromety analysis.

4)The stain we would be using is coomassie blue stain.

I hope i have answered your questions. Thanks!

MadTechs said...

Haha! You sure answered all my question crystal clear.

Thanks ya!
Quan Jun
TG02

Fluid collectors said...

hello benjamin. what are the different types of matrix compound? are you able to state the examples of matrix compound and the effect they do?

tg01 group 2 said...

Hello

Er, u didnt state ur name...

The matrix compound im using is alpha-cyano-4-hydroxycinnamic acid
and is a brownish grainy
material. The ionisation/dsroption proccess is triggered by a laser beam and is very strong. Hence, the matrix used (alpha-cyano-4-hydroxycinnamic acid) is to protect the biomolecule (periplasmic proteins) from being destroyed by direct laser beam and to facilitate vaporization and ionization.
However this compound matrix is not very effective as it leads to poor crysalisation also, but i have to use this for the ionisation process.

Other matrix compound can be 2,5-dihydroxy benzoic acid, 3,5-dimethoxy-4-hydroxycinnamic acid, 4-hydroxy-3-methoxycinnamic acid,
α-cyano-4-hydroxycinnamic acid and Picolinic acid.

Thanks for your questions

~immortals~ said...

hey ben

uhhhmmm... was curious, what's MALDI, MALDI plate? you said that its 'Matrix Assisted Laser Desorption Ionization', but what is it actually? what is it used for?

maya
tg02
grp 10

tg01 group 2 said...

Hey Maya

Maldi is used to analyse proteins that have undergone 2D gel electrophoresis and after excision (xcise). The Maldi plate is a medium in which to hold the proteins spots. *note: proteins are visualised as spots, not bands.

Thanks for your questions

tg01 group 2 said...

Hello Every1

I am very sorry... Periplasmic proteins ARE NOT found between the cell membrane. Periplasmic proteins are found between the periplasmic space which is between the inner and outer cell wall of a gram neagative bacteria. Please note: only gram neagtive bacteria has periplasm.


For further understanding of zymogram:

To run the zymogram, you will have to extract the periplasmic proteins. These periplasmic proteins contains periplasmic proteases. So when periplasmic proteins are seperated by sds page gel electrophoresis on a zymogram, only periplasmic proteases will digest the substrates on the zymogram and not the periplasmic proteins.

Sorry for the misunderstanding every1 ...

From: Ben
0606181F