iTRAQ Quantitative Proteomics Sample Selection

The basic principle of iTRAQ quantitative protein analysis applies labeling peptides with covalent bonding to the iTRAQ reagents on the terminal amino groups and lysine sides. After separation by high-performance liquid chromatography, primary mass spectrometry, and secondary mass spectrometry, proteins among the sample can be characterized and relatively quantified.

iTRAQ analysis is suitable for a wide range of sample sources, and virtually any sample collected can be screened using this technology in studying proteomics. Whether it is a search for functional proteins, mechanistic exploration, or screening of disease biomarkers, iTRAQ quantitative proteomics can translate samples into research results in the most convenient manner.

Theoretically, iTRAQ assays require at least 300 μg of protein per sample to achieve a successful experimental rate of usually less than 100%. Thus, the amount of prepared protein for each experiment should ideally be sufficient for two independent experiments. Moreover, collected samples may not always meet the needs of the investigation. Therefore, more thought needs to be given toward selected samples and how to handle them sufficiently.

There are various types of samples, including tissue, cells, blood (serum or plasma), cerebrospinal fluid, urine, bone, joint synovial fluid, etc., and collection methods vary between samples. In general, the followings need to be met:

Representative sample: The representativeness of the sample is directly related to the scientific significance of test results; thus, sampling plans need to be chosen carefully according to experimental purposes. There should be no contamination between the experimental samples and the control. When available, samples from the experimental and control groups should be kept consistently in terms of sampling time, site, and processing conditions. Otherwise, the overall credibility of experimental results may be affected. Body fluid samples need to be removed by stepwise centrifugation (or membrane) to eliminate interfering factors, including but not limited to cells, cell debris, and bacteria. The proteomes of different sample sources are varied, and even different cells of the same tissue sample can be varied significantly; thus, it is not comparable to quantify two samples that may be unrelated. As a result, it is important to consider a single variable principle in biological experiments when designing experiments to prepare samples.

Sample accuracy: Data of various representative samples must be recorded accurately. All samples must be collected, prepared, stored, and transported as required (low temperature and immediately), thus obtaining the final data according to the experimental design.

Sample timeliness: Sample quality is a key factor that affects experimental results in all types of testing experiments. Samples used for research should be collected, prepared, stored, and transported immediately to minimize sample collection time for experimentation. Particular attention should be paid to easily degradable samples such as the pancreas; and samples susceptible to microbial contamination, such as urine.

Cryogenic processing: After samples are collected, marked, and recorded, they should be snap-frozen in liquid nitrogen immediately and stored at -80°C. Do not put too much trust in the -80°C refrigerator. It is generally recommended that samples for proteomics studies should not be stored for more than a year, and long-term storage samples need to be qualitatively checked to determine stability prior to use.

It is recommended to reserve and back up samples simultaneously as they are taken to prevent partial degradation and delay in re-taking. Even if all samples are well qualified, backup samples can be used in different aspects of the experiment (e.g., quantitative validation, protein aspects, biochemical aspects, etc.).

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