Guide to Effective Sample Collection in Proteomics: Key Principles and Best Practices

Why Sample Collection Matters in Proteomics

Sample collection is a critical step in proteomics, as the quality and standardization of the samples directly affect the accuracy and reliability of experimental results. Therefore, in addition to meticulous experimental design, careful attention must be paid to the sample collection process. As the source of proteomics analysis, sample collection should adhere to the following principles.

Key Principles for Sample Collection in Proteomics

1. Representativeness and Consistency

To ensure reliable results, samples from both the experimental and control groups should be collected from the same location and under the same conditions, including time and handling processes. Any inconsistency could compromise the validity of the findings. For instance, the kidney is a heterogeneous organ with different cell types in the cortex and medulla, and the liver has distinct lobules and enzyme systems in its various zones. Sampling from non-representative or inconsistent sites can lead to skewed results.

2. Speed

Biological samples contain various enzymes that can rapidly degrade proteins at physiological temperatures. To minimize protein degradation, samples should be processed, stored, and transported as quickly as possible, reducing the time from collection to analysis to a minimum.

3. Low Temperature

Once collected, samples should be immediately processed at low temperatures, such as on ice or at 4°C. After separation, they should be stored in liquid nitrogen, on dry ice, or at -80°C to prevent protein degradation. Samples must remain below -80°C until use to maintain protein integrity.

4. Aliquoting

To prevent protein degradation caused by repeated freeze-thaw cycles, it is recommended to aliquot samples during collection. This approach allows for the use of appropriate volumes for different types of experiments, ensuring the reliability of data and accuracy of results.

Sample Collection Methods for Different Sample Types

After understanding the basic rules for sample collection, the next step is to gather the required samples for the experiment. Different types of biological samples have distinct collection methods. Below are the collection methods and recommendations for the most common sample types.

1. Animal Tissue

Animal tissue is a common sample type, and the key to its collection is to eliminate any potential blood contamination, as high-abundance proteins in blood can affect subsequent experiments. To ensure accuracy and homogeneity, it is crucial to avoid including interfering tissues, such as connective tissue. The collection standards and procedures are as follows:

1) Prepare cold storage tubes for sample packaging, labeling them with sample numbers using an oil-based marker.

2) Use animals that are similar in sex, age, and weight.

3) Accurately sample by removing connective and adipose tissue, cutting the tissue into small pieces, roughly the size of a soybean.

4) Quickly rinse the samples 3-5 times with pre-cooled saline or 1x PBS to remove blood and debris.

5) Blot dry with absorbent paper, transfer to storage tubes, and immediately snap-freeze in liquid nitrogen, then store at -80°C or ship on dry ice.

2. Clinical Samples

The biological reproducibility of clinical proteomics largely depends on sample selection and quality control. To ensure consistency, multiple factors should be considered, including the patient's age, sex, tumor location, morphology, clinical stage, type, size, and prior treatments (radiation, chemotherapy, immunotherapy, etc.), which significantly influence protein expression and modification levels. Given these considerations, obtaining eligible samples is crucial, requiring meticulous attention during collection and preservation. The general collection procedure is as follows:

1) Prepare cold storage tubes for sample packaging, labeling them with sample numbers using an oil-based marker.

2) Carefully excise the required tissue and cut it into small pieces, roughly the size of a soybean. Rinse the samples 3-5 times with pre-cooled saline or 1x PBS to remove blood and debris.

3) Place the tissue pieces in prepared storage tubes, quickly freeze them in liquid nitrogen, and store at -80°C or ship on dry ice.

A critical step is differentiating cancerous tissue from normal tissue. After surgery, the tumor tissue is identified as cancerous, while the tissue taken from the furthest edge of the tumor represents normal tissue. Histological examination (e.g., HE staining) is used to confirm the quality of the cancerous tissue, with the standard typically being that cancer cells must comprise over 60% of the tumor. Note that criteria may vary for different cancers. Understanding the distinctions among cancerous, adjacent, and normal tissues is essential when selecting controls based on the experimental objectives.

3. Cell Samples

Cell samples are widely used in experiments and can be categorized into adherent cells and suspension cells based on their growth characteristics. The collection methods for these two types of cells differ, as outlined below.

(1) Suspension Cells

1) Cultivate cells to a density of 2-5 × 10^5 cells/ml.

2) Transfer the culture medium containing the cells into a 15 ml centrifuge tube and centrifuge at 400-1000 g for 5-10 minutes.

3) Discard the supernatant and collect the cell pellet.

4) Resuspend the cells in 1 ml of pre-cooled 1× PBS and transfer them to a 1.5 ml centrifuge tube. Centrifuge at 1000 g for 5 minutes, discarding the supernatant, leaving no more than 10 µl of liquid.

5) Snap-freeze the washed cell pellet in liquid nitrogen and store at -80°C.

(2) Adherent Cells

1) After thawing, culture the cells for about five passages until they reach optimal growth conditions.

2) Remove the culture medium and add 1-2 ml of pre-cooled 1× PBS to the dish. Gently swirl to wash the cells and discard the supernatant. Repeat this wash step three times.

3) Gently scrape the cells into a centrifuge tube using a cell scraper. Seal the tube with parafilm, snap-freeze the cell pellet in liquid nitrogen, and store at -80°C.

An alternative method for collecting adherent cells is to use trypsin for digestion to detach the cells. However, this technique requires precise control of trypsin concentration and digestion time to avoid excessive digestion and protein degradation. Additionally, the cells must be washed with PBS to remove any residual trypsin to prevent contamination.

4. Microbial Samples

To avoid contamination from culture medium proteins, separate the microbes from the medium using centrifugation or other methods. Collect the microbial pellet and wash it three times with PBS. Resuspend the microbial pellet in PBS and transfer it to a 1.5 ml centrifuge tube. Centrifuge to remove the supernatant. For larger sample volumes, it is advisable to aliquot the samples into 50 µl portions, snap-freeze in liquid nitrogen, and store at -80°C.

5. Body Fluid Samples

Various types of body fluid samples are commonly used in laboratories, including serum, plasma, milk, saliva, and urine. The collection requirements differ based on the experimental objectives.

(1) Blood

• For Coagulation Factor Studies: Plasma is typically chosen for research on coagulation factors. Collect blood using an anticoagulant tube (usually EDTA). Gently invert the tube 180 degrees and mix 8-10 times to ensure uniformity between the blood and the additive. Centrifuge immediately at 4°C at ≤1300 g (adjusting for species) for 10 minutes. Transfer the plasma to a centrifuge tube, add protease inhibitors (if applicable), mix, and briefly centrifuge. Aliquot samples into 150 µl portions and store at -80°C. If there are no safety concerns, samples can be shipped; otherwise, consult technical personnel for appropriate inactivation methods.
• For Non-Coagulation Studies: Serum can be used instead. After collecting blood, allow it to clot naturally at 4°C for 30-45 minutes. Centrifuge at 4°C at ≤1300 g (adjusting for species) for 10 minutes and collect the supernatant. If there are no safety concerns, samples can be shipped; otherwise, consult technical personnel for appropriate inactivation methods.
• Avoid Hemolysis: It's essential to prevent hemolysis during the collection of both plasma and serum samples.

(2) Milk

Collect milk in sterile centrifuge tubes and store at -80°C. Use the acid precipitation method to remove casein by adjusting the pH to 4.6 with dilute hydrochloric acid, stirring to precipitate the casein.

(3) Saliva

Determine whether the focus is on proteins, cells, or microbial types present in saliva. Prior to collection, rinse the patient's mouth to avoid food residue contamination. Saliva secretion varies at different times, so it's crucial to plan the timing of collection. It is recommended to fast for at least 2 hours and collect samples between 9:00 AM and 12:00 PM. Centrifuge the saliva at 1000-2000 g or filter using a 0.22 µm membrane, aliquoting into three tubes of 0.1-1 ml each. Since saliva contains significant amounts of salivary proteases, collect samples quickly, snap-freeze them in liquid nitrogen, and store at -80°C.

(4) Urine

Collect urine samples without stabilizers. Centrifuge immediately at 4°C at 1500 rpm to remove cells and debris, then collect the supernatant. Perform low-speed centrifugation at 4°C at 14,000 rpm for 10 minutes to eliminate sediment. Concentrate the supernatant and aliquot into two centrifuge tubes, each containing about 1 ml. Snap-freeze in liquid nitrogen or dry ice and store at -80°C.

6. IP Samples

(1) Kit Method

Use the Pierce MS-Compatible Magnetic IP Kit (Protein A/G, ThermoFisher) for sample processing. Ensure the elution volume is not excessive. This method allows for subsequent enzymatic digestion and is recommended for label-free quantitative analysis.

(2) Acid Elution Method

Prepare the acid elution solution with 0.1 M glycine adjusted to pH 2.5 using HCl. Add approximately 40 µL of this solution to the centrifuged agarose bead-antigen-antibody complex and incubate for 2 hours. Centrifuge to collect the supernatant. If the wash buffer contains detergents, wash with a detergent-free buffer twice before elution. Keep the elution volume minimal.

If the lysis buffer contains Triton or SDS, the resulting protein solution requires acetone precipitation before enzymatic digestion, which may lead to some loss; thus, Method 1 is preferable.

(3) Proteins Bound to Beads Cannot Be Eluted

Centrifuge the sample to remove the supernatant, and send the protein bound to the beads for analysis, ensuring that all reagents used are compatible with mass spectrometry. Important notes: reagents used during IP should not contain NP40, Triton, or CHAPS, as these can affect detection results and damage the instrument. When sending protein samples, include a note on the buffer composition to facilitate subsequent experiments.

7. Paraffin-Embedded Tissues

(1) Paraffin-Embedded Tissues

Cut the samples into dimensions of 1.5 × 1.5 × 1.5 mm using appropriate instruments, minimizing excess paraffin. Collect at least six paraffin blocks per sample and ship at room temperature.

(2) Paraffin Sections

Provide a minimum of 10 sections and ship at room temperature.