Label Free Quantitative introduction
Label Free Quantitative analysis plays a crucial role in numerous fields. In the study of plant growth and development mechanisms, quantitative analysis of proteins in different stages of plant tissue can clearly reveal changes in protein expression, uncovering the molecular mechanisms that regulate plant growth. For example, studying the dynamic changes of various proteins during seed germination provides theoretical basis for optimizing crop cultivation.
In the study of plant stress resistance mechanism, the proteome of normal and stress (such as drought, high temperature) conditions can be compared to find out the key proteins responding to stress, which will help to cultivate more resistant plant varieties.
In the field of disease marker screening, biological samples from patients and healthy people are analyzed to find differentially expressed proteins as potential disease markers for early diagnosis.
In the study of drug action targets, Label Free quantitative service is used to observe the protein expression changes in cells or tissues before and after drug treatment, determine the key targets of drug action, and accelerate the development process of new drugs.
Characteristics of label-free sample preparation methods.
The importance of sample preparation
1. The effect on the accuracy of the experimental results
The sample preparation stage has a profound impact on the accuracy of experimental results. If sample preparation is improper, protein degradation can easily occur. For instance, if samples are not promptly and properly processed after collection or if storage temperatures are inappropriate, proteins may degrade under enzymatic action, leading to deviations in content determination. Moreover, impurity interference cannot be overlooked. If other impurities are mixed into the sample, they may compete with target peptides for mass spectrometry detection during LC-MS analysis, affecting the signal intensity of the peptides and making it difficult for the peak intensity to accurately reflect protein abundance, ultimately resulting in inaccurate quantification results. Additionally, poor sample homogeneity can also cause problems; uneven samples can lead to variations in protein content across different parts, thereby compromising the overall accuracy of experimental results and misleading subsequent research analyses.
2. The correlation with experimental reliability
Standard sample preparation is the cornerstone of enhancing experimental reliability and reproducibility. Proper procedures ensure consistent sample conditions across experiments, reducing fluctuations in results due to sample differences. For instance, strictly adhering to requirements for sample collection, processing, and storage can guarantee that proteins maintain their initial state across different batches, leading to stable and reliable signals in mass spectrometry analysis, thus ensuring high repeatability of experimental outcomes. Conversely, non-standard operations can introduce numerous issues. Inconsistent sampling sites may lead to inherent differences in protein expression, interfering with experimental results. Substandard storage conditions can alter protein structure and content, resulting in varied outcomes each time, making it impossible to draw reliable conclusions. This not only wastes time and resources but also risks deviating from research objectives, hindering scientific progress.
Sample type and requirements
1. Plant tissue samples
Plant tissue samples come in various types, including leaves, stems, roots, flowers, and fruits. For Label Free quantitative services, the required amount of different tissue samples varies. Generally, leaf samples should be provided at 100mg-200mg, stem and root tissues at 150mg-250mg, and flowers and fruits, due to their complex composition, typically require 200mg-300mg. The number of biological replicates is crucial; at least three biological replicates must be performed to ensure the reliability and scientific validity of the experimental results.
When preparing plant tissue samples, several basic principles must be followed. The principle of representativeness requires that the selected samples accurately reflect the overall characteristics of the subject. For example, if studying proteins related to photosynthesis in plants, mature leaves with active functions should be chosen. The principle of speed emphasizes that samples should be processed as soon as possible after collection to prevent proteins within tissues from degrading or changing due to prolonged storage. After collection, they can be immediately placed in liquid nitrogen for rapid freezing to maintain the original state of the proteins.
2. Microbial samples
When microbial (bacterial or fungal) samples are used for Label Free quantitation, specific requirements apply. In terms of sample volume, for bacteria, it is generally recommended to provide 1×10^8-1×10^9 cells; for fungi, typically 50mg-100mg of mycelium or spores are required. Biological replicates should also be performed at least three times to minimize experimental error and ensure the reliability of the results.
Special cases also require special attention. If the microorganisms under study are in a specific growth stage, such as bacteria in the logarithmic growth phase, it is essential to precisely control the culture conditions and timing to ensure that each sample collected has a consistent state. For some microorganisms that are difficult to cultivate, optimized cultivation methods or special sampling techniques may be necessary. If the samples contain significant impurities or metabolic products, which could affect protein extraction and mass spectrometry analysis, pre-treatment steps like centrifugation and filtration should be performed in advance to obtain pure microbial samples, laying a solid foundation for subsequent experiments.
3. Liquid samples
Liquid samples, such as root exudates, have unique requirements in Label Free quantitative services. The typical sample volume is 1-2 mL, which can be adjusted according to actual conditions. Key points for preparation include ensuring the integrity and stability of the sample. After collection, timely processing should be carried out to prevent degradation or denaturation of protein components.
Precautions must not be overlooked. Due to the complex composition of liquid samples, which may contain various impurities and small molecules, appropriate pretreatment such as ultrafiltration or centrifugation is necessary before protein extraction to remove impurities and enrich proteins. Additionally, attention should be paid to the storage conditions of the samples; they generally need to be stored at-80℃ to avoid repeated freeze-thaw cycles that could affect the structure and properties of proteins. During sample transportation, dry ice or liquid nitrogen should be used to maintain a low-temperature environment, ensuring that the sample quality is not compromised, thus guaranteeing the accuracy and reliability of experimental results.
Services You May Be Interested In:
Additional Resources:
Sample preparation process
1. protein extraction
Different types of samples require different protein extraction methods. For plant tissue samples, which are rich in polysaccharides and polyphenols among other secondary metabolites, protein extraction can be interfered with. Therefore, phenol extraction or modified TCA-acetonitrile precipitation is commonly used. During the process, tissues should be rapidly ground at low temperatures, and an extraction buffer containing protease inhibitors should be added to suppress protease activity and prevent protein degradation. When extracting animal tissue samples, a tissue homogenizer can be used to thoroughly grind the tissue, followed by centrifugation to obtain the protein supernatant. It is important to avoid excessive foaming during homogenization to prevent protein denaturation. For cell samples, cell lysis buffer can be directly added, and gentle stirring can lyse the cells and release proteins. Microbial samples require lysis first; Gram-positive bacteria can be treated with lysozyme, while Gram-negative bacteria often use ultrasonic disruption before protein extraction. Throughout this process, it is crucial to control temperature, pH, and other conditions to ensure the stability and integrity of the proteins.
2. Enzymatic purification
Protein enzymatic hydrolysis is the process of cutting proteins into smaller peptide segments, with trypsin being the most commonly used enzyme. It specifically cleaves peptide bonds at the carboxyl termini of lysine and arginine residues, producing peptides suitable for mass spectrometry analysis. During hydrolysis, protein samples are mixed with an appropriate amount of trypsin at a specific ratio and reacted for a certain period under suitable temperature (usually 37℃) and pH conditions to allow the enzyme to function fully. After hydrolysis, the peptides need to be purified. A common purification method is reversed-phase high-performance liquid chromatography (RP-HPLC), which separates peptides based on their distribution coefficients between the stationary phase and mobile phase. Through RP-HPLC, impurities, unreacted enzymes, and other contaminants can be removed from the hydrolysis products, enhancing the purity of the peptides. The purpose of purification is to obtain pure peptides, reduce interference from impurities on mass spectrometry analysis, improve the sensitivity and accuracy of mass spectrometry detection, and provide high-quality samples for subsequent Label Free quantification analysis.
3. Preparation before computer test
Before the on-machine testing, sample processing is crucial. First, sample labeling requirements must be met to ensure each sample has a clear and unique label. The label information should include key details such as the sample name, source, and processing conditions to prevent confusion among samples. In terms of storage conditions, purified samples are generally stored in-80℃ freezers to maintain peptide stability and avoid degradation or chemical changes. Transport conditions are also important; if samples need to be sent to other laboratories for testing, they should be transported using dry ice or liquid nitrogen to keep them at low temperatures throughout the journey. Additionally, the seal integrity of the packaging must be ensured to prevent leakage or contamination of the samples. Furthermore, a detailed sample information sheet should be prepared to record various sample details, including volume, concentration, and enzymatic conditions, so that test personnel can better understand the sample situation and make thorough preparations for the on-machine testing, ensuring the accuracy and reliability of the test results.
Common problems and solutions for sample preparation
1. Sample quality problems
The reasons for poor sample quality are varied. Sample contamination may stem from an unclean experimental environment, incomplete cleaning of equipment, or the introduction of other impurities or microorganisms, which can interfere with protein extraction and analysis. Sample degradation often results from untimely handling, such as failure to store samples at low temperatures promptly after collection, or temperature fluctuations during storage that allow protease activity to recover and cause protein degradation.
For solutions, if the sample is contaminated, it needs to be re-collected and the experimental environment must be strictly purified to ensure that all equipment is sterile. For degraded samples, the same procedure applies: re-collect the sample and optimize the processing steps. Preventive measures include operating in a clean environment such as an ultra-clean bench, and strictly sterilizing experimental equipment; immediately freeze the sample with liquid nitrogen or place it in an appropriate low-temperature environment after collection to avoid temperature fluctuations.
2. Sample marking and information filling
Clear and accurate sample labeling is crucial. Unclear labeling can easily lead to sample confusion in subsequent experiments, causing results to be muddled and making it impossible to accurately correlate the source and processing conditions of the samples. Inaccurate or incomplete information makes it difficult for testers to fully understand the samples, affecting the design of experimental protocols and interpretation of results.
Improper procedures can reduce the credibility of experimental results, even necessitating re-experiments. The solution is to use waterproof and low-temperature resistant labels, clearly marking key information with legible text; design detailed forms for filling out information, record simultaneously when collecting samples, and carefully verify after completion to ensure accuracy and reliability, thus providing a solid guarantee for the smooth conduct of experiments.
3. Sample preservation and transportation
Samples are prone to problems during storage and transportation. Improper temperature control is a common issue; high storage temperatures can lead to protein degradation and denaturation; malfunctions in refrigeration equipment during transport may cause samples to warm up and deteriorate. Additionally, inadequate packaging can expose samples to external air and moisture, further affecting their quality.
The correct storage method is to select an appropriate temperature based on the sample type; generally, protein samples should be stored at-80℃. During transportation, use a professional low-temperature transport box equipped with sufficient dry ice or liquid nitrogen to ensure the entire journey remains cold. Additionally, store the samples in sealed bags or sterile containers for multi-layer protection against leakage and contamination, ensuring that the sample quality is not compromised and providing reliable samples for experiments.
References
- Vowinckel J, Capuano F, Campbell K, Deery MJ, Lilley KS, Ralser M. The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics. F1000Res. 2013 Dec 13;2:272. doi: 10.12688/f1000research.2-272.v2. PMID: 24741437; PMCID: PMC3983906.
- Padula MP, Berry IJ, O Rourke MB, Raymond BB, Santos J, Djordjevic SP. A Comprehensive Guide for Performing Sample Preparation and Top-Down Protein Analysis. Proteomes. 2017 Apr 7;5(2):11. doi: 10.3390/proteomes5020011. PMID: 28387712; PMCID: PMC5489772.
