Note: This note was compiled from the Proteomics Online Class hosted by Crick College and Kang Yusheng in 2016-2017, and has been deleted. This course is taught by Mr. Li Qin from the School of Biology, China Agricultural University.
As users of mass spectrometers, how do we evaluate the performance of a mass spectrometer? In other words, how do we choose a mass spectrometer? The main performance parameters of the mass spectrometer are as follows. Let me explain to you in turn what these fancy parameter names mean.
The "official" definition is the amount of substance equivalent to three times the noise. We can understand that this is the concentration, or amount, of the lowest amount of compound that the mass spectrometer can detect. For compounds with lower values ??than this, this mass spectrometer will be powerless.
How do I know what the detection limit of my mass spectrometer is? Usually, we will use reserpine as a standard compound to determine the detection limit of the mass spectrometer. For example, when we inject 50 fg (fettg) reserpine into the mass spectrometer, if the signal-to-noise ratio we obtain can reach 100-1000, then it can be considered that the detection limit of this mass spectrometer is good. 50 fg (femtogram) of reserpine probably contains only tens of thousands of reserpine molecules. In other words, if it can detect compounds containing tens of thousands of small molecules, then the sensitivity of this mass spectrometer will be quite high. It's high. You can think that sensitivity and detection limit evaluate the same performance.
This performance parameter is also very important. It indicates within what concentration range there is a linear relationship between the signal detected by the mass spectrometer and the sample concentration. To put it simply, samples within this concentration range are more suitable for detection by this mass spectrometer. Samples above or below this concentration range need to be concentrated or diluted before they can be detected by this mass spectrometer.
Normally, the linear range of a mass spectrometer is 3-6 orders of magnitude, that is, within the range of 1,000 – 1,000,000. Most mass spectrometers are in the 1,000 – 10,000 range.
The reason why this parameter is important is that when the sample content we analyze has a very wide distribution, for example, some samples have only a few tens of μg/ml, while some samples can reach several mg/ml. . Within this relatively wide concentration range, if the linear range of the mass spectrometer is very good, we do not need to concentrate low-concentration samples, nor do we need to dilute high-concentration samples. We can directly inject samples, which can greatly reduce the complexity of sample preprocessing. degree, saving time and experimental steps.
These are two very important parameters. What we often call high-resolution mass spectrometry refers to extremely high resolution and extremely high mass accuracy. How to understand these two parameters? Let's first take a look at the picture below:
It is the mass difference between the two most recent mass spectrum peaks that the mass spectrometer can distinguish.
What does this mean? Suppose I have two mass spectrum peaks with the same intensity. When these two peaks are very close, under what circumstances can I clearly judge that these are two peaks instead of one? The basic criterion is that when the height of the overlapping portion of the two peaks does not exceed the peak height of any mass spectrum peak by 10, we consider these two separable peaks. On the contrary, if the overlap between the two peaks exceeds 10, they are considered inseparable. That is to say, when processing the mass spectrum, there is no way to process it as two peaks.
When two peaks achieve 10 baseline separation, we measure the half-peak width of any mass spectrum peak (that is, the peak width at half the peak height), and then divide the mass-to-charge ratio of any peak by Half maximum width, you can get the resolution.
At present, the resolution of high-resolution mass spectrometers can reach the order of 50,000-100,000, and the general quadrupole can reach 5,000-10,000.
So, how to reflect the advantages of high-resolution mass spectrometry? Take the picture on the right above as an example. When we use a low-resolution mass spectrometer to detect a certain substance, we can only get the outermost blue mass spectrum peak. As we continue to increase the resolution, we will slowly find that inside this mass spectrum peak , actually contains several small mass spectrum peaks. There is a very obvious difference between the mass-to-charge ratio obtained by high-resolution mass spectrometer and that obtained by low-resolution mass spectrometer. This is very important information for compound identification. If we calculate the mass-to-charge ratio incorrectly, it will be difficult for us to identify the correct protein.
The figure below can also intuitively tell us the advantages of high resolution in mass spectrometry detection.
For example, if we scan a compound with a resolution of 17,500, we will find that there is a very fat mass spectrum peak at the mass-to-charge ratio of 280.09 (the first spectrum (marked by a red circle), we may think that this is a compound, so we start to identify this compound. However, when we continue to improve the resolution of the mass spectrometer to a certain extent, we will find that these are actually two different peaks (the fourth spectrum is marked by a red circle).
In other words, using the mass-to-charge ratio obtained from low-resolution mass spectrometry to identify compounds actually yields incomplete information (not necessarily wrong), while through high-resolution mass spectrometry, we can We can obtain more comprehensive compound information and help us make correct judgments.
It refers to the difference between the mass-to-charge ratio measured by the mass spectrometer and its actual mass-to-charge ratio, divided by the product of its actual mass-to-charge ratio and 1,000,000. So it's in ppm (parts per million), which seems more convenient. The current mass accuracy of high-resolution mass spectrometers is within the range of 2-5 ppm.
So, how do we determine the actual resolution and mass accuracy of a mass spectrometer? Take an experimental data of Teacher Li Qin as an example:
For example, we select the peak with a mass-to-charge ratio of 511.6 and calculate its half-peak width as 0.012, so its resolution is 511.6 divided by 0.012 , the value obtained is 42,500, and the resolution given by the software is 48,700, which is very close.
Same example, let’s calculate mass accuracy. The measured mass-to-charge ratio is 511.5978, and the actual mass-to-charge ratio of this peak is 511.5995, so the mass deviation is calculated to be 3.3ppm, which means the error of this experiment is 3.3ppm. Such a mass deviation range is usually acceptable. of.
The importance of resolution may be easy for everyone to understand, but what impact does the level of mass accuracy have on compound identification? Let’s take reserpine as an example.
The reserpine molecule will have a mass spectrum peak at 609.28066 in the mass spectrum. When we use a single quadrupole to analyze reserpine, the mass accuracy of the single quadrupole is approximately 0.1 mass units (165 ppm). In other words, when reserpine is injected into a quadrupole mass spectrometer, the quadrupole mass spectrometer will tell us that the mass-to-charge ratio of this compound is approximately within the range of 609.2-609.4.
Then the problem arises! Within the range of 609.2-609.4, how many chemically possible compounds can we combine using the four elements C, H, O and N? The answer is: 209 species! In other words, if we want to judge whether this compound is reserpine, the possibility of getting the correct result is only 1/209!
As we continue to improve mass accuracy, there will be fewer and fewer possible compounds that can be combined. When the mass accuracy reaches 3 ppm, there are only 4 possible compounds. When it reaches 2 ppm, there are only two possible compounds left. At this time, if we judge whether the compound is reserpine, the accuracy will be much higher. This is why high-resolution mass spectrometry is so important for compound identification, as it can greatly reduce the number of candidate compounds and increase the success rate of identification.
It can be said that resolution and mass deviation evaluate the precision and accuracy of the mass spectrometer respectively. Just like when we are shooting targets, for example, if I can hit a point in the upper right corner every time, it means that the precision of target shooting is very high, but if my target is the bullseye, it means that the accuracy is relatively poor. In another situation, for example, I have been shooting many times, and the hit points are very scattered, one shot in the east and one in the west, but the average position is just on the bullseye. It can be considered that the quality accuracy is okay, but the precision is relatively poor. So what we want is for the mass spectrometer to be very precise and very accurate.
The high-resolution mass spectrometers we can currently use, whether they are QTOF or Orbitrap series, can achieve a resolution of more than 50,000, and can also achieve a mass accuracy of 2-3ppm. Therefore, children who are doing proteomics research today are much happier than before!
Earlier I shared with you several important parameters for evaluating a mass spectrometer. Next, we will make a rough summary of the performance of different mass spectrometers.
1. Quadrupole and ion trap: Their mass scanning range is limited, usually 10-4,000. Above 4,000, the quadrupole and ion trap can only be used for ion transmission and cannot be used for ion detection. Their resolution is usually 2,000-4,000, and better ion traps can reach 10,000-20,000. The scanning speed is not very fast, their advantage is that the price is very low, and the entire instrument can be made very small.
2. TOF: Its biggest advantage is that the measurable mass range can be theoretically infinitely large and infinitely small. If the ion to be detected has no mass, its flight time will be 0, so ions with a mass-to-charge ratio of 0 can be measured. In the same way, if the mass of an ion is infinite, its flight time is also infinite, and it can theoretically be detected. The resolution of TOF is 5,000-60,000, and the scanning speed is very fast. Its disadvantage is that TOF requires a very long ion runway, so the instrument will be very large.
3. FTICR: The advantage is that the resolution is very high, which can reach 1,000,000 or even higher. The disadvantage is that the scanning speed is relatively slow, and it requires a superconducting magnet, and the operating cost is very high, and The price of the FTICR mass spectrometer itself is also very high, usually above $1 million.
4. Orbitrap: Overcoming the shortcomings of FTICR that must use superconducting magnets, its resolution can reach 100,000 to 1,000,000, the scanning speed is not very fast, and the price is higher than that of FTICR Lower. It is patent protected and currently only produced by Thermo.
For proteomics research, our minimum requirements for mass spectrometry instrument performance are: resolution at least 40,000-50,000, mass accuracy should be better than 5ppm, and mass scan range should be within 100-3,000, the scanning speed is to obtain at least one high-resolution primary spectrum and ten high-resolution secondary spectra per second. If the above conditions are met, it will be considered as meeting the most basic requirements for proteomics.
The advantages and disadvantages of various mass spectrometers have been discussed above, so here we introduce the concept of tandem mass spectrometry: connecting the same or different mass spectrometers in series to achieve series or parallel operation. This has two purposes: to generate secondary fragment ions (we will talk about why secondary fragment ions are generated later), and to achieve the complementary advantages of different mass spectrometer performances.
We know that the performance of different mass spectrometers is different. For example, quadrupole mass spectrometry can achieve ion selection, but its resolution is relatively poor, while TOF cannot achieve ion selection, but its resolution is relatively high. So can we string together mass spectrometers with different performances and make them work together? We usually use tandem mass spectrometry or MS-MS to achieve this requirement. There are many ways to combine them:
The first one: Triple Quadrupole, or series quadruple, which is to connect three quadrupole in series. The main purpose of this is In order to realize the scanning of secondary mass spectrometry.
The second type: the quadrupole and time-of-flight mass spectrometer are connected in series, which is the Q-TOF we often hear. It is actually to improve the resolution of the secondary mass spectrometer.
The third type: the combination of Orbitrap and quadrupole, such as Orbitrap Fusion, or the combination of Orbitrap and ion trap, such as Orbitrap Elite, etc. This is such a combination.
First, let’s talk about how to obtain secondary fragment ions through a tandem mass spectrometer.
The above is a schematic diagram of a series quadruple rod structure. A tandem quad, or triple quad, is made up of three quads connected in series. Usually, the second quad is replaced by a sixth or eighth, but we still call it a quad. This quadrupole is not a mass selection system, but a collision cell, which is used to fragment ions.
When the series quadrupole works, the first quadrupole turns on the mass selection mode, which allows ions with a specific mass-to-charge ratio to pass through the mass spectrometer, while throwing away other ions. Drop (throw it onto the fourth-level pole, or throw it into the space of the fourth-level pole). Then, when specific ions are selected (called precursor ions), they enter the collision cell.
There is such a structure in the collision cell that there is a voltage difference between the inlet and the outlet. Usually the inlet voltage is higher than the outlet voltage. When the precursor ions come in, they will be accelerated by the voltage difference. Moreover, the collision cell will be filled with helium or nitrogen. When the ions are accelerated, they will collide and fragment with the helium or nitrogen molecules in the collision cell, forming some fragments, called fragment ions. Or product ions. These fragment ions will enter the third quadrupole and perform a second-level scan to obtain a second-level mass spectrum.
The picture below is a tandem quadrupole mass spectrometer. We can see that it is still a very compact structure.
Let’s take ractopamine as an example to see what changes will occur when its molecules pass through a tandem mass spectrometer, and what kind of spectrum will be obtained.
Ractopamine This is a veterinary drug.
Its molecular weight is 301.1672, and its structural formula is shown in the figure above. In the first picture, the measured mass-to-charge ratio is 302.1744. This is a scan of the first-level mass spectrum. There is a mass spectrum peak of ractopamine at 302.1744.
Then, we tell the mass spectrometer to pick out the ion at 302.1744 and set the CID (collision-induced dissociation) voltage to 10 volts, that is, add a 10V at the entrance and exit of the collision cell The voltage difference allows the ions to collide with a collision energy of 10V. After the collision, in the second picture, we see that the signal strength at 302 has become weaker, while the signal strengths at 284 and 164 have become stronger, and the 107, 121, and 136 signals that were not seen before have also appeared.
Next, we increase the collision voltage from 10V to 25V. After increasing, we will find that the signal at 302 completely disappears, indicating that the ion was originally selected in the first quadrupole , after a high-energy collision, it was completely fragmented into fragment ions such as 91, 107, 121, 136 and 164. So what are these fragment ions?
By analyzing the structure, we will find that they correspond to different fragment structures of ractopamine. For example, 164 actually corresponds to the right end of ractopamine, 136 corresponds to the left half, and so on.
By analyzing the chemical structure of the fragments, we can piece them together to form a complete ractopamine molecule. This is how we achieve structural identification of compounds through secondary mass spectra. While the actual identification process is often more complex and nerve-wracking, the above is just a simple example.
So for polypeptides, or for polypeptide fragments after proteolysis, we can go through the same process and analyze which fragments can be theoretically obtained from a polypeptide, and then compare it with the spectrum. Achieve identification of polypeptide sequences. This part will be discussed in detail later. Let's look at a simple example first, as shown below:
For example, if we have a peptide segment like this in the upper left corner, theoretically we can get various b-y ions marked in gray. By analyzing the mass spectrum, we can get Find the corresponding fragment ions (the ones marked in red in the table on the right are fragment ions that can be found in the mass spectrum), and by assembling this information, we can know what the sequence of the polypeptide is.
The above uses the triple quadrupole as an example to share with you how the tandem mass spectrometer obtains secondary fragment ions and secondary spectra. Then, some other tandem mass spectrometers have a similar process.
Q-TOF is actually very similar to a tandem quadrupole, except that it replaces the third quadrupole with a time-of-flight mass spectrometer. That is, a quadrupole, followed by a collision cell, and then a time-of-flight mass spectrometer. In order to increase the flight distance, we will make the ions turn a corner and then fly back. This is called reflection mode flight, which allows the ions to fly farther in a shorter space.
The picture below is a Q-TOF mass spectrometer, produced by Bruker. The length of its flight tube can reach 3.6 meters, and the ion travel time is 7.2 meters. You can pay attention to this number, and it will be mentioned again when talking about vacuum degree later.
The Orbitrap series is more complicated than ordinary tandem mass spectrometers. You can feel it through the schematic diagram below.
There are several tandem mass spectrometers in this series, such as the Q Exactive mass spectrometer. Its Q1 is also a quadrupole, Q2 is a collision cell, and Q3 is replaced by an Orbitrap.
Another example is the Orbitrap Elite. Its Q1 is an ion trap, Q2 is a collision cell, and Q3 is an Orbitrap. In other words, there is no quadrupole in the Orbitrap Elite. It uses an ion trap. Replaced the quadruple pole.
There is also the Orbitrap Fusion (see the picture below), which is a combination of three mass spectrometers mixed together. Its first stage is a quadrupole, the second stage is an ion trap, and the third stage is an ion trap. The stage is an Orbitrap, and it also has a collision pool. The overall structure is a very complex one. Its characteristic is that the Orbitrap and the ion trap can scan simultaneously.
In a general mass spectrometer, two mass detection quantities cannot be scanned at the same time. One can only be used as a mass detection function and the other as a filter. The ion trap and Orbitrap in Orbitrap Fusion can scan at the same time, that is to say, it is a parallel structure, not just in series, so its scanning speed will be faster and its performance will be better. Fusion's resolution can reach 240,000 – 960,000.
I have shared several commonly used mass spectrometers above. Taking Q-TOF as an example, let’s learn the basic structure of a mass spectrometer.
For a mass spectrometer, the core part is the mass analyzer, which consists of two parts, the mass filter and the mass detector that we introduced in detail earlier. All other parts of the mass spectrometer serve this core part.
In addition to this core component, the mass spectrometer also requires the following assistance:
Vacuum system: Why is a vacuum system needed? We know that a mass spectrometer is an instrument that detects the mass-to-charge ratio of gaseous ions. When a gaseous ion flies in the air, it will collide with air molecules, and its charge may be destroyed and become a For uncharged gaseous molecules, the mass spectrometer can no longer measure its mass-to-charge ratio. Therefore, we hope that the gaseous ions we obtain can exist stably in the mass spectrometer, so the mass spectrometer needs a vacuum system to allow the ions to fly stably without interference from other air molecules.
Vacuum systems usually need to have two levels. The first level is low vacuum, which is provided by a mechanical pump or an oil pump. It can provide a pressure environment of about 1-3 mbar, which is one thousandth of an atmosphere. The purpose of low vacuum is to provide a backup pressure environment for high vacuum. High vacuum is provided by a turbine pump, and its vacuum level is -1E-5~-1E-10 mbar. In such a vacuum environment, the air molecules are basically pumped out.
You may want to ask, why are vacuum conditions of this order of magnitude required?
Let’s first introduce a concept called the mean free path of ions. It means how long an ion will fly in a vacuum environment before it encounters the next air molecule. This determines how long the ions can remain stable in a vacuum.
Taking the tandem quadrupole as an example, the tandem quadrupole mass spectrometer is about 1 meter long, so we hope that the ions will not encounter other air molecules while flying 1 meter.
So for the series four-stage rod, as long as the vacuum is maintained to ensure that no air molecules are encountered within a distance of 1 meter. So a series quadrupole usually only requires a vacuum of -1E5 mbar.
For Q-TOF, the flight distance of ions is about 5-7 meters (do you still remember? The flight distance of 7 meters was specifically mentioned when introducing Q-TOF earlier). Compared with The flight distance of the tandem quadrupole is nearly one data level longer, so the vacuum degree required by the Q-TOF mass spectrometer is about -1E-6 ~ -1E-7 mbar to ensure that the ions will not Collision with other air molecules.
For the Orbitrap mass spectrometer, the ions can fly inside for 1 second and fly a very long distance, so Orbitrap requires a vacuum degree of -1E-10 mbar.
Ion source system: We need to introduce the sample from the non-ionizing environment of external atmospheric pressure into the mass spectrometer and turn it into a gaseous ion, so an ion source is needed to realize this function.
Computer system: realizes the control of the mass spectrometer and the collection of data.
Gas system: gas supply and exhaust gas treatment (nitrogen, argon)
Power supply: UPS uninterruptible power supply system
Plus core component mass analyzer , the above are the six systems that make up the mass spectrometer. We will also discuss the structure, use and maintenance of each part later.
A mass spectrometer with these six components installed can be represented by the following schematic diagram. Usually, the mass analyzer and high vacuum turbine pump are installed in a large box. This module is called the main unit, and the low vacuum pump (oil pump) is placed outside the main unit, because this part will produce a lot of vibration, noise and The heat needs to be kept separate to prevent vibration from affecting the mass spectrometer. There will be an ion source in front of the mass spectrometer and an exhaust port on the side. The exhaust gas generated by the mass spectrometer and pump will be discharged to the outdoors through this exhaust pipe. In particular, exhaust gases produced by pumps are often carcinogenic, so exhaust is particularly important.
The above discusses how to evaluate the performance of a mass spectrometer, how a tandem mass spectrometer works, and the six components that make up a mass spectrometer. The next article will talk about the composition of a liquid chromatograph and the working principle of liquid mass spectrometry equipment.