Protein mass spectrometry analysis is a powerful technique used in the field of biology to investigate and characterize proteins. This method plays a crucial role in unraveling the intricate details of biological systems by providing insights into the composition, structure, and function of proteins. The process involves ionizing protein molecules, separating them based on their mass-to-charge ratio, and then detecting and analyzing the resulting mass spectrum.
In the realm of proteomics, protein mass spectrometry is particularly valuable for identifying and quantifying proteins, studying post-translational modifications, and elucidating protein-protein interactions. It enables researchers to gain a comprehensive understanding of cellular processes, disease mechanisms, and biomolecular dynamics.
Protein mass spectrometry has revolutionized the field of biology, enabling researchers to delve deeper into the complexity of biological systems and paving the way for advancements in personalized medicine, drug discovery, and understanding the molecular basis of various diseases.
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Principles to Follow in Sample Preparation
Principle of Representativeness
The representativeness of samples is crucial for the scientific significance of experimental results. Therefore, careful consideration should be given to the selection of sampling schemes based on the experimental objectives. Tissue samples from pathological specimens should not contain normal tissues, and normal tissue samples should be free of pathological tissues. Whenever possible, efforts should be made to maintain consistency in sampling time, location, and processing conditions between experimental and control groups. Failure to do so may compromise the credibility of experimental results.
Principle of Accuracy
Various characteristic data of representative samples must be accurately recorded and collected, prepared, stored, and transported as required (e.g., at low temperatures and promptly). This ensures that experiments and data processing are conducted correctly according to the experimental design.
Principle of Promptness
Sample quality is a critical factor influencing the results of proteomic experiments. Therefore, samples used in proteomic research should be handled as promptly as possible during the collection, preparation, storage, and transportation processes, minimizing the time from sample collection to experimentation.
Principle of Low Temperature
After collection, samples should be placed in liquid nitrogen (preferred) within 10 minutes. If conditions are too rudimentary, an alternative option is storage in dry ice or a -80°C freezer (not preferred). It is crucial to ensure that samples remain at or below -70°C at all times before the experiment to prevent the degradation of modified proteins and proteins with low abundance expression.
Sample Quantity Recommendations
Table 1. Minimum amount for routine sample proteomics
Table 2. Minimum amount of proteome in exosome-like body fluid samples
Notes:
a) For Label-free and DIA experiments, it is recommended to submit 50% of the sample quantity required for TMT/iTraq.
b) For Phosphorylation TMT experiments, it is advised to submit twice the amount of samples required for regular TMT, and the addition of a phosphatase inhibitor is strongly recommended before conducting phosphorylation modification proteomics experiments.
c) For N-Glycosylation Label-free experiments, it is recommended to submit twice the amount of samples required for regular TMT.
d) For other modifications such as acetylation and ubiquitination in proteomics experiments, it is recommended to submit ten times the amount of samples required for regular TMT.
e) For animal brain tissues, pituitary glands, and fluid exosomes, there may be fewer differential proteins, which is considered normal.
f) Clients providing proteins should include SDS-PAGE electrophoresis images to assess protein extraction effectiveness and provide information on the reagent composition of the protein solution.
g) It is recommended to store samples in high-quality, imported 2 mL screw-capped cryotubes. If only centrifuge tubes are available in the laboratory, seal the tube openings with parafilm.
h) Declare in advance if the sample contains toxic or corrosive substances. For pathogenic microorganisms or tissues with invasive properties, samples must be inactivated before submission. Indicate the treatment process and the formula of the reagents used for already processed samples. Specify any special characteristics of the sample material, such as thick capsules or susceptibility to oxidation.
i) For Label-free and TMT experiments targeting serum and plasma samples, additional charges apply for the removal of high-abundance proteins. However, DIA experiments do not require the removal of high-abundance proteins from serum and plasma.
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Methods of Sample Preparation
Susp ension Cells
Collect well-growing cell suspension from a culture dish or flask. Centrifuge at 200-1000g for 5-10 minutes, discard the supernatant, and collect the cell pellet.
Resuspend the cell pellet in 1×PBS prepared with enzyme-free water. Centrifuge at 200g for 5 minutes, discard the PBS, and wash twice.
Transfer to screw-capped cryotubes suitable for -192°C, freeze rapidly in liquid nitrogen for 3-4 hours, then transfer to a -80°C freezer for long-term storage or ship on dry ice.
Adherent Cells
Retrieve adherent cells from the incubator, ensuring good growth under a microscope. Discard the culture medium and invert the culture dish on absorbent paper to remove residual medium.
Add 1×PBS prepared with enzyme-free water, gently shake for 1 minute to wash cells, discard PBS, and wash twice to remove culture medium.
Place the culture dish on ice, add pre-chilled PBS, and scrape cells quickly with a clean cell scraper on one side of the dish. Tilt the dish on ice to allow buffer flow to one side. Pipette the lysate into a pre-chilled centrifuge tube. Centrifuge to remove the supernatant.
Transfer to screw-capped cryotubes suitable for -192°C, freeze rapidly in liquid nitrogen for 3-4 hours, then transfer to a -80°C freezer for long-term storage or ship on dry ice.
Notes:
a. Do not ship cells directly without proper freezing, as ice crystals may easily puncture cells during freezing, leading to protein degradation.
b. Avoid using trypsin for cell digestion, as it may digest membrane proteins, causing cell rupture and release of various enzymes.
Animal Tissue/Clinical Tissue Collection
Obtain fresh tissue, keeping the volume small, ideally with dimensions not exceeding 0.5cm in length, width, and height for ease of handling. Total weight of routine samples should be no less than 100mg.
Remove non-relevant tissue types (e.g., connective tissue, fat) and, for clinical pathological tissue, accurately distinguish between lesion and normal tissue, removing surrounding normal tissue if necessary.
Quickly wash the tissue surface with pre-chilled enzyme-free water-prepared 1×PBS or physiological saline, and absorb any liquid on the surface.
Place the prepared tissue in pre-chilled, enzyme-free, screw-capped cryotubes labeled with numbers, freeze rapidly in liquid nitrogen for 3-4 hours, then store at -80°C for long-term preservation before protein extraction. Avoid repeated freeze-thaw cycles. Ship on dry ice.
Notes:
Hard tissues like cartilage should be cut into small pieces around 1cm before rapid freezing in liquid nitrogen. Tough tissues like hair should be rinsed with 2% SDS, pH=7.8, 50nM sodium phosphate buffer to remove contaminants before drying and rapid freezing in liquid nitrogen.
Plant Tissue Collection
Collect fresh tissue from plants, with a recommended total sample weight not less than 1g. For larger tissues, attempt to cut them into small pieces.
(Optional for some plant samples) Quickly wash the tissue surface with pre-chilled enzyme-free water-prepared 1×PBS or physiological saline and absorb any liquid on the surface. Cut into suitable sizes with scissors (ideally no longer than 10cm unless in special circumstances).
Place the prepared tissue in pre-chilled, enzyme-free, screw-capped cryotubes labeled with names, or wrap in foil with marked names, freeze rapidly in liquid nitrogen for 3-4 hours, then transfer to a -80°C freezer for long-term storage before protein extraction. Avoid repeated freeze-thaw cycles. Ship on dry ice. If not rapidly frozen with liquid nitrogen within 10 minutes after removal, samples may undergo protein denaturation. Do not store samples directly at -80°C, as low-activity metabolites and catalytic enzymes may still react.
Notes:
Bark, roots, leaves, etc., are advised to be washed with PBS to remove soil or contaminants before rapid freezing in liquid nitrogen. During the flowering period, collect pollen, remove anthers, and check pollen morphology and purity under an optical microscope before rapid freezing in liquid nitrogen and storage.
Fluid Samples (Including Exosomes)
a. Serum Samples (Exosome-like)
Recommend using imported medical serum tubes or vacuum blood collection tubes to collect whole blood or screw-capped tubes to collect whole blood samples.
Gently invert the tube ten times, immediately place the whole blood upright at 4°C or in a cooler.
Centrifuge at 1800g for 10 minutes to separate the upper serum sample (serum separation should be done within 1 hour, following the instructions for serum tubes or the customer's laboratory serum separation procedure).
Transfer the serum to a 1.5mL centrifuge tube, centrifuge at 13000g for 2 minutes.
Transfer the supernatant to cryotubes with capacities ranging from 200μL to 1.5mL suitable for -192°C, with each sample having a minimum volume of ≥100μL (0.1mL). Freeze rapidly in liquid nitrogen for 3-4 hours and store at -80°C, ship on dry ice.
b. Plasma Samples (Exosome-like)
Recommend using purple EDTA anticoagulant tubes to collect whole blood samples, and prohibit the use of heparin anticoagulant tubes for whole blood collection.
Gently invert the tube ten times, immediately place the whole blood upright at 4°C or in a cooler.
Centrifuge at 1200g for 10 minutes to separate the upper plasma sample (plasma separation should be done within 1 hour, following the instructions for anticoagulant tubes or the customer's laboratory plasma separation procedure).
Transfer the plasma to a 1.5mL centrifuge tube, centrifuge at 13000g for 2 minutes.
Transfer the supernatant to cryotubes with capacities ranging from 200μL to 1.5mL suitable for -192°C, with each sample having a minimum volume of ≥100μL (0.1mL). Freeze rapidly in liquid nitrogen for 3-4 hours and store at -80°C, ship on dry ice.
Notes:
• The separation of serum and plasma should strictly follow the operating instructions. The procedure should be completed within 1 hour to avoid hemolysis, and after separation, repeated freeze-thaw cycles should be avoided.
• The principle for the quantity of serum/plasma sent for analysis is the more, the better. If the customer has a limited amount of serum/plasma, send as much as available, with a minimum recommendation of 100μL.
• Whole blood from serum/plasma separation should not be frozen, and samples with mixed blood cells should be discarded as much as possible and are no longer usable.
c. Cell Supernatant, Urine, Saliva, Amniotic Fluid, Cerebrospinal Fluid (Exosome-like)
Collect cell supernatant and fresh urine (≥50mL), saliva, amniotic fluid, and cerebrospinal fluid (≥2-5mL), ensuring no hemolysis (blood proteins may contaminate sample proteins). Place them upright at 4°C or in a cooler, transfer to a 15-50 mL centrifuge tube. The following steps should be completed within 2 hours after sample collection to prevent protein degradation.
Centrifuge at 1000 rpm for about 10 minutes at 4°C, remove cells and debris.
Carefully pour the supernatant into a new 15-50 mL centrifuge tube (be cautious not to transfer the precipitate), centrifuge at 2500 rpm for about 10 minutes at 4°C to remove remaining debris and cells.
Transfer the supernatant to a new cryotube suitable for -192°C, freeze rapidly in liquid nitrogen for 3-4 hours (optional), seal tightly. Store at -80°C, ship on dry ice.
Notes:
If there are too many fragments in the cell supernatant, low-speed centrifugation should be carried out multiple times to remove the fragments thoroughly. For urine collection, a light diet is recommended the day before. For saliva collection, fasting and abstaining from smoking and alcohol for at least 2 hours are required. Samples should be collected between 9:30 AM and 11:30 AM. Rinse the mouth with distilled water, spit into a sterile sputum cup (maintained in a water bath), avoid coughing, collect saliva for about 30 minutes, centrifuge at 4°C, 10000 rpm for 10 minutes, collect the supernatant, and filter through a 0.22μm membrane for sterilization.
Non-pathogenic Yeasts, Mold Fungi, Bacteria, Bacteriophage (Microorganisms)
The wet weight of the sample should be ≥200mg. Observe the growth status of yeast or bacteria under a microscope and collect logarithmic-phase yeast or bacteria.
Transfer an appropriate volume of yeast liquid to a 2 mL imported screw-capped centrifuge tube, centrifuge at 14000g for 4 minutes at 4°C.
Discard the culture medium, quickly place the yeast cell precipitate in a -192°C ultra-low-temperature screw-capped cryotube, freeze rapidly in liquid nitrogen for 3-4 hours, store at -80°C, and ship on dry ice.
Note:
Steps two and three are optional, and one to two additional washes with PBS are recommended to remove residual medium.
Fungi (e.g., Mushrooms, Black Fungus)
The wet weight of the sample should be ≥2g. Weigh the fungi and divide them into multiple tubes.
Place the weighed fungi in a pre-cooled mortar, add liquid nitrogen, grind the fungi thoroughly, transfer to -192°C ultra-low-temperature screw-capped cryotubes, store at -80°C, and ship on dry ice.
Protein Solution
Provide an adequate amount of whole protein samples according to experimental requirements (protein precipitation is recommended). Protein quantity should be ≥500µg (precipitate volume approximately 20-30µL). If it is necessary to send the sample in protein liquid form, please inform about the buffer components, such as whether it contains SDS, enzyme inhibitors, etc., and the protein quantity should be ≥500 µg. Store at -80°C, ship on dry ice.
Organelle Proteins, Membrane Proteins
For identification and analysis of organelle proteins and membrane proteins, we currently do not undertake the separation and extraction of organelles, cell membranes, and cell walls. Customers need to perform this extraction themselves (common methods involve density gradient centrifugation). The volume of the organelle or cell membrane precipitate sent should be ≥50μL. If proteins have already been extracted, please refer to 2.8 Protein Solution.
