iTRAQ on the tip of the tongue: four principles for the selection of proteomics research

The doctor who doesn't want to be a cook is not a good scientist - Nicholas Huang Huang

China's food culture is profound and profound, and "China on the tip of the tongue" shows us the perfect food-eating nation. A successful dish, the selection of ingredients has very strict standards. In the field of basic medical research, experiment and cooking also have something in common. If you are lazy during cooking, you can use the soup cooked with noodles to cook, and the taste of the oysters is greatly reduced. The experiment is also the same, one is one, two is two, it is necessary to be precise and prudent, and it is not allowed to be lazy and flexible. Stealing work and reducing materials for a while, the data is difficult to repeat, while the cottage virus saves money, the doctor delays crying into a dog.

The most important thing to cook is the quality of the raw materials. Before you put it in your mouth, you never know that the taste of the dish is not correct, but the fresh and high-quality ingredients can at least improve the success rate of this dish. After all, the stinky fish can never be pulled out. Fresh soup. The same is true for proteomics research. A qualified sample without pollution or degradation is a prerequisite for finding a differential protein! In this issue, Huang Bo introduces the sample selection of a hard dish in the high-throughput screening technology of quantitative proteomics, iTRAQ combined with tandem mass spectrometry.

The basic principle of iTRAQ quantitative analysis of proteins is that the iTRAQ reagent can be covalently linked with amino acid terminal amino groups and lysine side chain amino groups to complete the labeling of peptides by high performance liquid chromatography, first-order mass spectrometry and second-order mass spectrometry. Mass spectrometry can complete the qualitative and relative quantification of proteins in the sample. In theory , the amount of protein required for each sample in the iTRAQ experiment is at least 300ug . However, in fact, anyone who has done the experiment should understand that the success rate of all experiments is not 100% . Especially for iTRAQ, the process is complicated and stable to the instrument. Sexually dependent experiments, so it is conservative to ensure that the amount of protein prepared before each experiment is best to satisfy two independent experiments, which is one. The second is the sample preparation stage. From the perspective of experimental technology, iTRAQ experiments can be performed as long as the protein can be extracted from the sample . But for the purpose of the experiment, do the samples we collect really meet the requirements? Not necessarily! Especially for clinically collected samples.

One of the advantages of iTRAQ is that the sample source is extensive. Almost all collected samples can be used to screen proteomes, whether it is searching for functional proteins, exploring mechanisms, or screening Biomarker for disease. The result is to avoid these precious samples sleeping in the refrigerator.

Samples are available in a variety of forms, tissues, cells, blood (serum or plasma), cerebrospinal fluid, urine, and synovial fluids. Different sample collection methods vary, requiring a targeted protocol. In general, the following conditions must be met. A few:

Representation principle: The representativeness of sampling is related to whether the experimental results are scientifically meaningful, so you should carefully choose your sampling plan according to the purpose of the experiment. There should be no pollution between the experimental and control samples. When there are conditions, the samples of the experimental group and the control group should be as consistent as possible in terms of time, location, and processing conditions. Otherwise, the reliability of the experimental results may be affected. . Body fluid samples need to be removed by stepwise centrifugation (or membrane) to remove interference factors such as cells, cell debris and bacteria. Another very important point is that the proteomes of different sample sources are different, even the difference between different cells in the same tissue will be very large. The quantification between the two samples is not comparable, so the design experiment is prepared. The sample must be considered in the single variable principle of biological experiments.

Accuracy principle: The various characteristic data of representative samples must be accurately recorded, collected, prepared, stored, and transported as required (low temperature, rapid), and finally experimentally and data processed according to the experimental design. In addition, the accumulation of detailed clinical data (including pathological data, prognostic follow-up data, etc.) is an important factor in ensuring the clinical significance of data analysis results.

Timeliness principle: Sample quality is the key factor affecting the experimental results in various testing experiments. Therefore, the samples used for research should be as fast as possible in the process of collection, preparation, storage and transportation, and the sample should be shortened as much as possible. The time at which the experiment was collected. Pay particular attention to such easily degradable samples as the pancreas, as well as samples of urine that are susceptible to microbial contamination.

Low temperature principle: After the sample is collected, mark and record it, quickly freeze it in liquid nitrogen, store it in a refrigerator at -80 °C, and send it to dry ice. The whole process should avoid repeated freezing and thawing of the sample to prevent protein degradation. But don't rely too much on the -80° refrigerator. Even if the temperature of the refrigerator fluctuates smoothly, the samples stored in it will still slowly degrade. It is generally recommended that the samples of proteomics research should not be stored for more than one year. Samples stored for a long time need to be determined by quality inspection. Can still be used.

In order to ensure the smoothness of the experiment, it is strongly recommended that when sampling, it should be backed up at the same time, one copy, one copy, in case some samples are degraded, and the time is re-acquired. Even if the samples are all qualified, the backed up samples can be used for other experiments (such as quantitative verification, protein, biochemical experiments, etc.). There are two types of backups, one is strict backup, that is, the sample is taken down and divided into two, and the two samples are basically of the same nature. The other is a non-strict backup, that is, a biologically duplicated sample. Such a backup sample is less homogenous than the former.

Finally, for the iTRAQ experiment, different experimental design, different sample collection methods, Huang Bo is equipped with corresponding private goods, welcome everyone to poke WeChat private chat.

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