Sunday, July 22, 2007

Lab Techniques

Topic: MALDI TOF (matrix-assisted laser desorption/ionization-time of flight)

As mentioned earlier in Ming Boon's post, the MALDI TOF is used in the analysis of the proteins that were initially separated through 2D gel electrophoresis and excised using Xcise. The peptides are then spotted, together with the matrix compound, on a 384-well MALDI plate (sample plate). Unlike Xcise, there was no formal training for us on the use of the 4800 Plus MALDI TOF/TOF Analyzer.

In this post, i will try my best to explain more on the principles of MALDI TOF.

The samples are analyzed in a two-step process. During the first analysis, the samples (peptides) are ionized using laser. When the laser hits the spot (contains the peptides mixed with the matrix) on the sample plate, it will cause the matrix to be ionized. The ionized matrix will transfer its ion to the sample. This ionizes the peptides. Another beam of laser is flashed onto the spot with a high voltage applied to the sample plate. This will cause the ionized peptides to accelerate out of the plate and into the flight tube.

The ions are accelerated with the same potential (energy) and form up at a fixed initial point and time. (It is like a race where all the runners form up at the starting line.) The ions are then allowed to drift down the flight tube. They will separate base on their mass to charge ratio, the lighter ions will have a higher velocity while the heavier ions will have a lower velocity. The time taken for an ion to travel from the start to the end, where the detector is, through the flight tube is measured i.e the time of flight (TOF). The m/z value is then obtained. That is for the first part of the analysis.

The second part of the analysis will involve the fragmentation of ions analyzed earlier. The form of fragmentation used in this analyzer is known as Collision Induced Dissociation (CID). The ions (parent ions) are selected to enter the collision cell based on the parameters set earlier. Inert gas molecules are introduced into the collision cell at a certain pressure. The ions are then selected by the precursor ion selector and transmitted into the cell. The gas molecules and ions will collide with one another and during the collision, there will be transfer of energy which causes the fragmentation of the parent ion. This will give rise to daughter ions which are then subjected to analysis. Like the parent ion, the daughter ions will be analyzed by measuring the time of flight and the m/z value is then obtained. [This also explains for the double TOF seen in the name of the analyzer.]

During the analysis, the measurements (m/z values) are shown as peaks. At various peaks, it is checked if it meets certain criteria like resolution, which will impact the following steps. When a parent ion is fragmented, it gives rise to daughter ions.These ions can be detected from the peaks. They can be b-ions (charge remains on the N-terminal of the peptide) or y-ions (charge remains on the C-terminal of the peptide). B-ions comprise of an amino acid (eg lysine) and a proton (hydrogen ion) while y-ions are made up of an amino acid and water. The amino acid sequence is deduced by the difference in mass between consecutive ions which corresponds to the mass of the individual amino acid.

That will be all for my post. I apologise if my post is unclear or kinda choppy in terms of the content. I will try my best to answer any queries that you guys may have aite? Till then enjoy your SIP & see all of you on Friday! Cheers!

Posted by Shahirah Bibi, TG01, 0503174E.


5 comments:

phuiyuen said...

hello :)

so the purpose of this is to analyse proteins? all kinds of proteins can be processed?

does it involved a very long duration of time?

that will be all.

phuiyuen, TG02

phuiyuen said...

hello :)

so the purpose of this is to analyse proteins? all kinds of proteins can be processed?

does it involved a very long duration of time?

that will be all.

phuiyuen, TG02

J.A.M.M.Y.S said...

Hi phuiyuen

thanks for the question. yup it is to analyse the proteins and pretty much all proteins can be analysed.

The duration really depends on the number of samples that u are running. It can take anywhere between a few hours to one whole day.

hope i ve answered ur question. see u dring the next campus discussion! :)

Shahirah TG01.

Star team said...

Hey Shahirah,

What kind of QC do you do for MALDI TOF?

Randall
TG02
0503272G

J.A.M.M.Y.S said...

Hi Randall,

i'm not too sure if there's QC for this machine. I will find the answer and get back to you aite? :)

Shahirah TG01