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MALDI-TOF Mass Spectrometry Explained

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.