Proteomics sample preparation: Choosing the right extraction methods
Common types of proteomics samples include cell samples, animal tissue samples, plant tissue samples, paraffin-embedded tissue samples, fungal samples, serum and other body fluid samples, IP samples, and more. Each type of sample requires a tailored preprocessing method.
1. Cells
Cell samples are relatively easy to process, and various common methods are available, including direct lysis on ice with lysis buffer, repeated freeze-thaw cycles, ultrasonic treatment after adding lysis buffer, and high-temperature SDS boiling extraction. Typically, ultrasonic treatment with lysis buffer is chosen for extracting cellular proteins, which is similar to the process for animal tissue samples. This method usually requires a cell quantity of around 10^6 cells.
However, newer technological methods demand much smaller cell quantities, such as micro-sample proteomics, which allows proteomics experiments with 10 to 1000 cells. With the advancement of proteomics technology, it is even possible to perform proteomic analysis on a single cell, known as single-cell proteomics.
2. Animal Tissues
The processing methods described here are suitable for more common animal tissue samples, including heart, liver, spleen, lung, kidney, muscle, and brain. Tissues such as skin, hair, and bone require special treatment and are not included.
Typically, animal tissue samples contain blood, which, if not removed, can introduce high-abundance blood proteins into the sample and interfere with subsequent mass spectrometry identification. Therefore, tissue samples should be washed with PBS to remove blood. To prevent protein degradation during washing, this process should be carried out on ice and ideally completed before the samples are frozen to avoid repeated freeze-thaw cycles that can degrade proteins.
After washing, tissues can be ground using liquid nitrogen or homogenized. If the sample quantity is large, a tissue grinder may be used. Once the tissue is broken down, lysis buffer (either 8M urea or RIPA buffer) is added. Sonication is then used to aid in further lysing the tissue. After sonication, the solution should appear clear. If the solution remains cloudy, it may indicate insufficient lysis due to an inadequate amount of lysis buffer.
Following lysis, centrifugation is required to remove any unlysed tissue and impurities such as connective tissue. Typically, this is done at 4°C at 15,000 g for 10 minutes, and the supernatant is collected. Samples with high fat content may present a thick layer of fat on top, with possible unlysed tissue precipitate below. Repeated centrifugation and collection of the supernatant may be necessary to obtain a cleaner protein sample.
3. Plant Tissues
For plant tissue samples, the main challenges are breaking down the cell wall and removing chlorophyll. Since plant cells have cell walls, unlike animal cells, standard homogenization is insufficient to fully break down the cell walls. Therefore, liquid nitrogen grinding is typically used to break down plant tissues and cells.
After grinding the plant tissue, a strong lysis buffer is usually chosen to further break down plant tissue cells and extract proteins. Commonly used lysis buffers include phenol extraction reagent or SDS lysis buffer. Due to the high fiber content and thick cell walls in plant samples, ultrasonic treatment, shaking, and various combinations of methods are employed to extract proteins.
After extracting proteins from plant tissue samples, pigments must also be addressed. Plant pigments are small organic molecules that, if not removed thoroughly during preprocessing, can affect subsequent protein quantification and be difficult to remove during desalting. This can lead to strong background peaks that interfere with mass spectrometry detection and impact detection depth. Pigments are typically removed through acetone or TCA precipitation, which first precipitates the protein and then washes away pigments from the protein surface, as pigments dissolve in acetone. Finally, the protein is resolubilized with 8M urea solution, and the supernatant is collected after centrifugation to obtain a protein solution.
It is important to note that there are several special types of plant samples, such as seeds and fruits. Some seeds contain high oil content and require pre-treatment to remove lipids. Seeds with high sugar content necessitate the use of an appropriate lysis buffer. Certain fruits also contain high water content, resulting in low protein yields; thus, extraction may require increasing the sample amount or freeze-drying the sample to remove moisture before protein extraction.
4. Bodily Liquids
There are many types of body fluid samples, but for this discussion, we will focus on blood as an example to introduce preprocessing methods for blood samples. Blood contains large amounts of high-abundance proteins, with the top 14 high-abundance proteins making up over 95% of total blood proteins. Since mass spectrometry is an ion saturation detector, signals from low-abundance ions can easily be suppressed by high-abundance ions, making them undetectable.
In previous blood proteomics experiments, the conventional method was to use kits to remove high-abundance proteins from the blood before conducting mass spectrometry. However, with advancements in mass spectrometry and preprocessing methods, blood proteomics samples can now be divided into two types:
1. Blood without removal of high-abundance proteins: Blood samples are directly preprocessed to obtain peptides, which are then analyzed using high-performance mass spectrometry instruments and the DIA data acquisition mode. This approach reduces interference from high-abundance protein removal and lowers experimental error. However, detection depth remains limited due to the presence of high-abundance proteins.
2. Blood with high-abundance proteins removed: There are many commercial kits available for removing high-abundance proteins from blood. These methods generally fall into two categories:
1) Non-antibody-based methods: A representative product is Bio-Rad's Proteominer kit. These kits specifically bind to low-abundance proteins in samples, allowing excess high-abundance proteins to be removed. This approach can identify a significant number of proteins. For example, the timsTOF Pro2 mass spectrometer in DIA data acquisition mode can identify approximately 1,500 proteins. This non-antibody approach is not limited by species or sample type, offers good reproducibility, and is cost-effective.
2) Antibody-based methods: Available products include Agilent's MARS column, Thermo's Pierce Albumin/IgG kit, and SIGMA's Human IgY14 and SuperMix Columns. These products use specific antibodies to remove high-abundance proteins from blood before mass spectrometry analysis. This approach significantly enhances identification depth in blood proteomics. Given the specificity of the antibodies for high-abundance proteins, these methods offer stable and reproducible results and are widely accepted in scientific literature.
In addition to the aforementioned four types of high-abundance removal kits, there is another kit available on the market that utilizes magnetic beads to enrich low-abundance proteins. Testing has demonstrated that when blood samples are processed with this kit and analyzed using the timsTOF Pro2 mass spectrometer's DIA data acquisition mode, approximately 3,800 proteins can be identified within one hour. Moreover, the data shows excellent repeatability and stability, achieving a reproducibility rate of over 0.98. These kits have significantly enhanced the depth of identification in serum proteomics, and their cost is only slightly higher than that of Bio-Rad's Proteominer kit. In the future, they may emerge as the predominant option for enriching low-abundance proteins in blood samples on the market.
Read more:
· A Guide to Protein Database Selection
· MetwareBio Launches Proteomics Services
· What is Isoelectric Points of Amino Acids: Essential Calculations and Practical Applications
· Comparison and Application of Proteomic Technologies
· Demystifying Proteomics Research Strategies and Content in a Single Read
· Optimal Protein Database Selection: Insights from Experimental Data
