Common sample types of proteomics research are roughly divided into the following categories: cell samples, animal tissue samples, plant tissue samples, paraffin-embedded tissue samples, bacterial samples, serum samples, IP samples and so on. Each type of sample has a slightly different pretreatment method, next, we choose a few types of samples to talk about the problem of sample protein extraction in detail:
Cellular Samples
Cellular samples are relatively easy to handle, and there are many common processing methods, including adding lysate directly on ice lysis, repeated freezing and thawing lysis, adding lysate after ultrasonication, SDS high temperature boiling extraction, etc. Generally speaking, we will choose to add lysate to the cellular samples.
Generally speaking, we will choose to add lysate and then sonicate to extract cellular proteins, and the experimental procedure is similar to that of animal tissues. Usually, we require the cell volume to be of the order of 106. Of course, there are some new technologies that require a very small amount of cells, for example, at this stage, we often refer to the micro-sample proteomics, proteomics experiments can be carried out on 10-1000 cells; with the development of proteomics technology, and even a cell for proteomics analysis, such as single-cell proteomics and so on.
Animal Tissue
The processing methods mentioned here are only suitable for relatively conventional animal tissue samples, including heart, liver, spleen, lung, kidney, muscle, brain and other tissues; skin, hair, bone tissue, etc. are not included in this category and require special processing methods. Generally speaking, animal tissue samples contain blood, and if not removed, high-abundance blood proteins will be introduced into the sample, which will affect subsequent mass spectrometry identification. In pretreatment, it is necessary to wash the tissue sample with PBS. To avoid protein degradation during the cleaning process, it is generally operated on ice. Laboratories with conditions should complete the cleaning before the sample is frozen, which can avoid the protein degradation caused by repeated freezing and thawing in subsequent cleaning.
After cleaning, the tissue is crushed by liquid nitrogen grinding or homogenization. If the sample volume is relatively large, a tissue grinder can be used for sample grinding. After tissue crushing, add lysis buffer, and the lysis buffer generally chooses 8M urea lysis buffer or RIPA lysis buffer. Then use ultrasound to further assist in lysis. After the ultrasound is completed, the solution is generally transparent. If the solution is turbid, it means that the lysis buffer is added too little and the lysis is insufficient.
After the lysis is completed, it is necessary to centrifuge to remove undissolved tissue components and other impurities, such as connective tissue, etc. Generally, 4℃ 15000g centrifugation for 10min is used, and then the supernatant is taken. Special attention should be paid to the fact that some samples have a higher fat content, and a large layer of fat can be seen on the top during extraction, and there may be undissolved tissue precipitation below. At this time, the method of repeatedly centrifuging and taking the supernatant can be used to obtain a protein liquid sample with higher cleanliness.
Plant Tissue
For plant tissue samples, the difficulty lies in cell wall fragmentation and chlorophyll removal. Since plant cells have more cell walls than animal cells, general homogenization simply can't handle the cell walls and there is no way to break them sufficiently, so liquid nitrogen grinding is usually used to break tissues and cells for plant tissue samples.
After the grinding of the sample tissue is completed, we will use a lysis solution with strong lysis ability to further break the plant tissue cells to extract the proteins, and the commonly used lysis solutions are phenol extraction reagent or SDS lysis solution. Since the plant samples have more fibrous tissues and the plant cell walls are thicker, we have to use ultrasound, shaking, and a combination of methods to extract the proteins.
After taking care of the protein extraction of the plant tissue sample, we need to deal with the pigments. Plant pigment is a kind of small molecule organic matter, if it is not cleanly removed in the pretreatment, it will affect the subsequent protein quantification, and in the next enzyme desalting, it is also more difficult to remove, and finally cause strong background peaks, which will interfere with mass spectrometry detection and affect the detection depth. Generally the pigment is removed by acetone or TCA precipitation, firstly using acetone or TCA can make the protein precipitation, and then using the principle of pigment dissolved in acetone to clean the pigment on the surface of the protein.
Finally, the protein that precipitated is dissolved again in an 8M urea solution, and the resulting solution is centrifuged to obtain the protein solution.
Different plant samples, including seeds and fruits, have unique characteristics. Some seeds have high oil content, so it is necessary to remove the oil by other methods in advance; some seeds have high sugar content, so it is necessary to choose the appropriate lysis solution; some fruits also have high water content, so the protein yield is relatively low, so it is necessary to appropriately increase the amount of samples, or freeze-drying of samples after the extraction of proteins.
Body Fluid Samples
There are more types of body fluid samples, here we mainly take serum as an example to introduce the pretreatment method of serum samples. Serum samples contain a large number of high abundance proteins, the first 14 high abundance proteins account for more than 95% of the total serum proteins. Whereas mass spectrometry is an ion-saturated detector, the signals of low-abundance ions are easily suppressed by the high-abundance ones, and thus cannot be detected.
In previous serum proteomics experiments, the routine method is to remove the high abundance proteins in the blood with a kit and then perform mass spectrometry. Nowadays, with the development of mass spectrometers and the innovation of pretreatment methods, blood proteomics can be roughly divided into two categories:
Category 1: without removing high abundance proteins in blood. Blood samples are directly pre-processed to obtain peptides, and then proteomics experiments are performed with the help of high-performance mass spectrometry instruments and DIA data acquisition modes. The advantage of this is that it can reduce the interference of removing high peaks on serum samples, and reduce the experimental error to a certain extent; however, due to the influence of high abundance, the detection depth is still limited, and the current timsTFO Pro2 mass spectrometer can identify about 800 proteins with a single needle under the DIA data acquisition mode.
DIA workflow (LCGC North America.Volume 35, Issue 10, pg 756–759)
Category 2: Remove blood high abundance methods. There are more kits on the market for removing blood high abundance, and their principles can be roughly categorized into two groups:
1. Antibody-independent methods. This type of kit can affinity the low abundance proteins in the sample with high specificity, and those excess high abundance proteins which are not bound to the binding site will be removed. The final number of proteins that can be identified after processing by this method is also relatively high, in terms of the number of proteins identified timsTOF Pro2 Mass Spectrometer instrument in DIA data acquisition mode can identify about 1500 or so proteins. This method is non-antibody based, it is not limited by species or sample type, has better reproducibility and is less expensive.
2. Antibody-dependent methods. These products remove highly abundant proteins from blood through the use of specific antibodies, followed by mass spectrometry. Antibody-dependent methods can greatly improve the depth of proteomics identification. Due to the targeted removal of high-abundance proteins in blood, the experiments exhibit high stability and reproducibility, resulting in relatively high article acceptance rates. However, the method's disadvantage lies in its expensiveness, costliness, and difficulty to store.
In addition to the above 4 kinds except high abundance kits, there is another kind of magnetic bead method enrichment of low abundance protein kits on the market at present, through the test, with timsTFOPro2 mass spectrometer, the DIA data acquisition mode about 1 hour can be identified to about 3,800 proteins, and the data reproducibility and stability is very good, the experimental repeatability of performance reaches more than 0.98 (as shown in the following figure). Utilizing this type of kit brings the depth of identification of serum proteomics to a new level and lays the foundation for a better search for biomarkers. Moreover, the cost is only about higher than that of Bio-Rad's Proteominer kit, and the author believes that the magnetic bead method for serum low abundance protein enrichment kit will surely shine.
After the proteins are extracted by various methods, this is not the end of the matter, because we need to carry out quality control on the extracted proteins to confirm whether enough proteins are successfully extracted, whether the protein amount meets the standard and so on. General quality control is divided into two parts:
Content determination, to determine the extracted protein concentration and protein amount, and according to the amount of extracted protein to calculate the protein yield, to assess the effect of protein extraction. Commonly used protein quantification methods are BCA and Bradford method, need to pay attention to the incompatibility of quantitative reagents, if the sample has SDS, Triton and other decontaminants, do not use the Bradford method to determine the protein concentration, you can use the BCA method; if the sample is added to the reducing agent, EDTA concentration greater than 2mM or more, do not use the BCA method to Determine the protein; if both decontaminants and reducing agents are contained, the protein needs to be precipitated with acetone and re-solubilized with a buffer that does not contain the above substances before protein quantification.
SDS-PAGE involves taking equal amounts of protein samples and analyzing the effects of protein extraction and the accuracy of protein quantification using strips. During the process, various situations may arise that require countermeasures. The table below outlines a few possible scenarios and provides corresponding solutions.
| Problems with SDS-PAGE | Possible causes of sample | Recommended sample size |
|---|---|---|
| Similar conditions but different shades | Inaccurate quantification | Re-quantification |
| Large differences in bands between duplicate samples | Insufficient protein extraction | Re-extract protein |
| Few protein bands | Insufficient extraction or sample itself | If it is not due to the sample, then the lysate should be changed or the extraction conditions should be intensified. |
| Fuzzy bands | Excessive impurities in the protein solution or slight degradation of the sample. | Rerun SDS-PAGE after precipitation. |
| No bands | Protein degradation | Reprepare sample |
At this point, we have introduced the problems of protein extraction and quality control in preprocessing proteomic samples. The involved sample types are complex, and the encountered issues may be diverse. In summary, adherence to several elements is necessary, including attention to and compliance with:
Protein Sample Extraction Tips
In summary, adherence to several elements is necessary, including attention to and compliance with:
- Simple processing methods should be used to extract proteins as much as possible while ensuring effective protein extraction.
- High-abundance proteins pose a significant challenge to mass spectrometry detection, and their introduction in experiments should be minimized as much as possible.
- Decontaminants like SDS must be used with caution while extracting proteins as they are not compatible with subsequent mass spectrometry and must be removed before analysis. Additionally, consistent formatting and citation styles must be followed for an academic writing quality.
- Proper storage and preservation of protein solutions are necessary after extraction to avoid degradation due to repeated freezing and thawing. It is crucial to avoid biased language and adhere to formal grammar and precise vocabulary.
- The complexity of samples necessitates flexible application of various pre-treatment methods.
References
- GebreyesusSofani Tafesse,Siyal Asad Ali,Kitata Reta Birhanu et al. Streamlinedsingle-cell proteomics by an integrated microfluidic chip anddata-independent acquisition mass spectrometry. Nat Commun, 2022,13: 37.
- WangWei-Qing,Jensen Ole Nørregaard,Møller Ian Max et al. Evaluation ofsample preparation methods for mass spectrometry-based proteomicanalysis of barley leaves. Plant Methods, 2018, 14: 72.
- SoniRajesh Kumar,High-Throughput Plasma Proteomic Profiling. MethodsMol Biol, 2022, 2546: 411-420.
- ShehadulIslam M, Aryasomayajula A, Selvaganapathy PR. A Review on Macroscaleand Microscale Cell Lysis Methods. Micromachines (Basel). 2017 Mar8;8(3):83.
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