MALDI-TOF Mass Spectrometry Explained
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- Опубліковано 27 лип 2024
- 0:00-0:23 | What is MALDI-TOF?
0:23-1:00 | What are the different parts of MALDI-TOF?
1:00-2:35 | How does MALDI-TOF work?
2:35-3:24 | MALDI explained
3:24-5:28 | How are proteins analyzed using MALDI-TOF?
5:28-6:50 | Why is MALDI-TOF useful?
MALDI-TOF is an acronym which stands for Matrix-Assisted Laser Desorption Ionization Time-Of-Flight and is a type of mass spectrometry, meaning that it measures the mass of the components within the sample.
In general, all mass spectrometers consist of 3 major components: 1. an ionization source, 2. an analyzer and 3. a detector. In addition they also have an inlet for sample loading and a computer for data analysis. Mass spectrometers are usually ionized by either electrospray ionization (ESI), which I will cover in another vide OR matrix-assisted laser desorption (MALDI) which is of course what the MALDI-TOF uses. We’ll come back to this MALDI part of the device in a second. Regardless of the method for ionization the point of this part of the process is to ensure that the sample molecules become electrically charged. Then the mass analyzer separates them based on their mass-to-charge ratio or m/z. In the case of MALDI-TOF the sample gets separated based on the time it takes its constituent parts to fly through the time-of-flight or “drift” region of the detector. Since the sample has been ionized it can be accelerated by a high-voltage current and fly through the tube before it strikes the detector. The time needed for each molecule to reach the detector depends on its mass, meaning that smaller molecules will reach the detector faster than larger one. This has to do the fact that each particle is accelerated at the same electric potential AND the fact that kinetic energy is half the mass times the velocity squared. $Ek = 1/2 mv^2$. Since the kinetic energy and the electric potential energy equal each other $Ek = qV$, or $qV = 1/2 mv^2$ the smaller the particle is, the faster it must go! The detector records the number of events within a specific time period and uses the information of calibration standards to determine what the sample consists of. The pattern analysis performed is quite complex and reminds of that used in artificial intelligence work rather than what we usually see in analyzers in the field of proteomics.
Okay, that is the TOF or time-of-flight part, now let us return to the MALDI or matrix-assisted laser desorption part of the device. The matrix acts as a sort of protective shield by absorbing the laser light and transferring it to the sample molecules. Direct exposure to the sample would ruin it because it would cause peptide fragmentation. The matrix and sample is mixed at a ratio of 1000-10 000 parts matrix to 1 part sample. This mixture is then spotted onto a stainless steel, and allowed to dry into crystals. Then with the energy from the laser the sample can desorb into a positively charged gaseous phase with very little accidental fragmentation.
Let us now look closer at how the protein is actually identified. To identify a protein based on its total molecular weight alone would be difficult to say the least. Therefore the protein gets digested and we can analyze multiple smaller peaks instead of one big. This gives us multiple datapoints or clues that we can compare to existing data to identify the correct protein. To make this easier, consider a combination lock. Say you have the code 100 but this feels to easy for someone to accidentally crack. So instead you put on 3 smaller looks that add up to 100 but that one have to solve simultaneously in order for the lock to open. Say you put the code 24, 65, and 11. The likelihood you would put these three numbers into the correct locks by accident is much smaller. In our case it is similar, the more specific we can make the results from the MALDI-TOF the less likely we are to identify the wrong protein.
Finally why is the MALDI-TOF useful? Well, as a mass spectrometer it can be used whenever we are investigating the components that make up any molecule. Recently there has also been a growing interest if the MALDI-TOF could be used in identifying specific bacteria in routine health care checks as well. Some studies such as the one by *Seng, P. et al. (2009)* suggested that it is a more time efficient and cost effective method than the ones currently used and could replace Gram staining and biochemical identification.
Please ask any questions you may have!
Can you please explain DESI technique?
Why is kinetic energy = electric potential in this concept ?
Great Video! Love your channel! I have some minor corrections to your video:
1) TOF: detected m/z value is dependent on BOTH the molecular mass ("size") and the CHARGE of the analyte. it is not only dependent on the "size" as you stated. A peptide with a mass of 1 kDa, for instance, can be detected at ~ 1kDa m/z if it bears 1 charge, but also at 500 Da m/z if it bears 2 charges, and at 333 Da m/z if it bears 3 charges.
2) you should not use the term "size" when referring to the molecular mass of a molecule. These are 2 different physical properties.
Thanks! :)
Great points! Thank you so much! I've been considering to make an updated version to this video since I could make a much nicer version today so when I do I'll make sure to include your corrections!👍
Thanks for the video! Very clear and simple explanation
Thank you so much for those nice words Octavio!😇
Very helpful! Thanks!
Excellent! Glad to hear it! I also have videos about MALDI and TOF separately if you wish to know more about either one of these two techniques😇👍
Great explanation
Thankyou 🤟
Great that I could help!
thx! it was very helpful for my presentation
I am happy to hear that!👍
Thank you great video
Glad to hear that you found it helpful! Let me know if you have any questions or suggestions for other topics to cover!👍
Thanks a million for this fantastic educational video. I have two suggestions, I guess it would be much better if you use some pictures from real instruments which are used in laboratories, and secondly, if you produce a series of videos on using the common analyser software to teach the whole PMF process, it would be great.
Great suggestions! I'll see what I can do!
THX!!!! Great explanation!
Happy I was able to help!
Thank you!!
Happy I could help!!😇👍
What exactly is the difference between MALDI-TOF and MALDI? Whitout measuring the TOF how does MALDI analyze the samples?
Excellent question! The MALDI part is ONLY responsible for ionizing the sample while the TOF is ONLY responsible for measuring the ionized sample.
In other words, we can use another ionization method such as for instance electrospray ionization (ESI), which I cover in another video, instead of MALDI. In the same way, we can use another method instead of TOF to measure our sample.
You can think of this as two different parts of one larger device that can be changed out depending on what samples you are measuring and so forth... First "real-life" metaphor that comes to mind would be in gaming, where you can either use a PC, Playstation or an Xbox and plug this device into a desktop screen, or some type of TV... Not the best analogy but hopefully that answers your question!
@@biotechlucas4126 perfectly explained, thank you very much!!
@@noraspeiser1865 Glad to hear that, thanks for commenting!!
once you have "digested" the proteins and splitted them into parts of different molecular weights, how do you know which parts belong to the same protein in order to have an accurate identification of it?
This depends. Sometimes you do your best to isolate the protein as much as possible before you use MALDI-TOF.
However, often you might compare the results to existing libraries of mass spectra in order to try and find a match.
Does that help at all?
Thanks
Happy to help!😇
Hello this was a great video explaining how MALDI-TOF works! I am currently using the MALDI-TOF on some peptides I'm making and I had a question. The peptides we are making has a +4 net charge but on the maldi we usually see +1 charge mass (m/z) on the spectra and not the +4 charged (m/z) mass. Can you explain this? Thank you!
How does that show up exactly? I mean how do you know that you're seeing +1 charge rather than +4 charge?🤔
I've only analyzed things that had a +1 charge myself I'm afraid🙈😅
@@biotechlucas4126
Hi thanks for responding! I know that it is the +1 charge because that is the predicted mass of my peptide. My peptide has a mass of 1154 g/mol and that is the peak that I see on the maldi-tof. I am just confused because the peptide has charged amino acid residues that should be fully protonated with a net +4 charge when it is on the plate with the matrix.
Nice ❤
I'm happy you liked it!😇🙏
Excellent teaching🎉Can you add a video on NMR spectroscopy?
A short video series on different aspects of NMR will be the next thing I do! Promise! :D
Hi I'm currently trying to analyse both positive and negative MALDITOF spectra for some lanthanide complexes I synthesised to try and determine the molecular weight and hence structure of my products, but I'm really struggling to understand how to interpret the spectra. I would really appreciate any help you can offer.
If you mail me some of the spectra at biotechlucas@gmail.com I can take a look😇👍
Also if you know what matrix you're using and the standard used for calibration that would help as well🙏
Make video on illumina sequencing
Here you go😇
ua-cam.com/video/1kUNGeW-KKQ/v-deo.htmlsi=XdXbyBtO-CfHVxZj
can you describe how to build one? thanks.
Interesting question! What is the context if I may ask?😇 Do you mean theoretically or how these are built by different companies?
So MALDI ionization technique is same as Chemical Ionization (CI) technique in GC mode?
Not entirely sure what you mean. Chemical Ionization is also an ionization technique true but they are different in terms of how they are carried out and how well they preserve the sample.
Can you go more in-depth about MALDI?
Absolutely, might do it this week or the next week at the latest!