Q&A of Proteomics

A: In principle, as long as you can extract proteins, you can do proteomic testing. The number of proteins measured depends on the database and the concentration of proteins in the sample, generally the larger the database, the more proteins can be identified. It is also related to the type of sample; usually, tissue cells can identify more proteins than body fluid samples.

A: Different samples will be sent with different requirements, and providing them according to the collection method will ensure that the amount is sufficient for the experiment. We do not need to count or weigh the samples accurately, but we will determine the protein concentration before the project, and then we will process and test different samples with different protein amounts, but we need to make sure that the sample size of each sample meets the experimental requirements.

A: For routine proteomic assays, we perform protein concentration measurements to determine if the amount of protein is sufficient for the subsequent assay. We will also run SDS-PAGE to see if the protein is degraded and if the bands are obvious. If the amount of protein is not enough or if there is an abnormality in the gel, we will give feedback to the customer and communicate the solution in time.

A: Serum, plasma, cerebrospinal fluid and other body fluid samples have 97%~99% of high abundance proteins, including albumin, immunoglobulin, IgG, IgA, antitrypsin, fibrinogen, transferrin, etc. The presence of high abundance proteins can interfere with the detection of low abundance proteins, which are often biomarkers or drug targets of greater concern, and it is routinely recommended to remove high abundance proteins before testing.

A: You can cut the concerned part of the strip for testing, or you can test the whole lane of a single sample. If the whole lane is tested, it is recommended to divide it into at least two copies, one for heavy and light chains and one for the other, which can increase the depth of testing. The gel strips are shipped in ice packs.

A: Both can be used. Plasma contains fibrinogen, and the abundance of protein detected in serum is higher compared to plasma, and some trace proteins may be detected in serum. However, the procedure of serum is more complicated than that of plasma, which may lead to human error.

A: Label free and TMT&ITRAQ belong to data-dependent acquisition mode (DDA), which usually selects TOP N parent ion peptides for secondary fragmentation detection, while DIA products belong to data non-dependent mass spectrometry acquisition mode, where all parent ions can enter secondary fragmentation and the number of proteins that can be characterized may be higher, depending on the library building data;

Compared to labeled proteomes, neither DIA nor label-free is limited by the number of samples. The reproducibility of DIA is higher compared to label-free technology. This technique is recommended for relatively large sample sizes.

When the number of samples is small, if there is a large difference between samples and you need to check the presence or absence of information about the protein, it is recommended to do the label-free product.

If the number of samples is small and you want to perform a deeper detection and more accurate qualitative quantification, labeled quantitative products are recommended.

A: Currently, there are two main types of labeling kits: TMT/iTRAQ. The kits can label up to 16 samples at a time, and if there are more than 16 cases, the calibration between batches can be achieved by bridging the samples.

A: It is common to use the protein database of the species on the UniProt website as the search file. If the database of the species is small or not available, you can choose the following three solutions: (1) expand the species hierarchy and choose a higher level or larger species database; (2) choose a close species database; (3) use the transcriptome sequencing results as the database. Some individual species have specific database sites that can also be used for library search analysis.

A: Experimental steps: Firstly, we obtain biological samples, then we perform protein extraction, quantification, SDS-PAGE running gel quality control, reductive alkylation, enzymatic digestion, peptide fragmentation, (labeling + subfractionation), online detection, library search software, and data analysis.

Instruments: Q Exactive™ HF-X, Orbitrap Fusion™, Orbitrap Exploris™ 480, etc. are mainly used.

A: Data analysis: It is divided into three parts: basic data analysis, high-level data analysis, and personalized data analysis. The basic analysis mainly includes differentially expressed protein screening, hierarchical cluster analysis, COG annotation analysis, subcellular localization analysis, GO annotation enrichment analysis, KEGG annotation analysis, PPI analysis, etc.

A: In principle, the more biological replicates the better, to exclude individual differences, the more accurate the screened differential proteins, and the higher the validation success rate. Considering the cost, the need for statistical analysis and the possibility of questioning by subsequent editors, it is recommended to have more than ten replicates per group for clinical samples and at least three replicates per group for samples from other sources.

A: Regular biological samples such as tissues, cells and body fluids need to be cryopreserved and sent on dry ice. Glue stick samples can be sent in ice packs.

A: The results of proteomics assays are compared to known database proteins and cannot predict unknown proteins. If unknown proteins need to be detected, other methods such as sequencing can be used. If the database does not contain the protein of interest, then it cannot be compared to it. The protein sequence of interest can be added to the database as a library search file for analysis.

A: First of all, it may be because the database is relatively small, resulting in fewer results. You can choose to expand the hierarchy or select the more studied database of closely related species or model species to search the database for analysis. Secondly, look at the lower gel map to determine whether the sample itself has fewer bands and whether there are high abundance proteins, the presence of high abundance proteins will affect the number of detections.

A: Due to in vivo or in vitro factors, the same protein is modified, sheared or degraded in different forms, resulting in protein bands with different molecular weights as seen in the gel electrophoresis image. However, these proteins are identified by mass spectrometry and point to the same, full-length, unmodified protein theoretical sequence in the database. Therefore, the molecular weight of the protein in the gel electrophoresis map (actual molecular weight) is different from the molecular weight of the identified protein (theoretical molecular weight).

A: The conditions of differential protein screening are mainly combined with the p-value and FC-value of the t-test, generally labeled products are screened according to FC>1.2 or FC<0.83, p<0.05; non-labeled products are screened according to FC>1.5 or FC<0.67, p<0.05. The actual process will also be combined with the test results to relax or card strict, generally control within 20% of the test results, 5-10% is better.

A: Conventional protein validation methods include WB, ELISA, PRM, and if the number of proteins of interest is small and there are commercial kits or antibodies available, it is recommended to choose ELISA or WB methods for validation, which are more mature. If the number of proteins of interest is large and there are no commercial antibodies, it is recommended to use the PRM method. Antibody preparation is also a good choice when funds are available.

A: Western blot is a highly sensitive assay that amplifies the target protein signal by many steps and (except for specific binding) is almost independent of the background protein abundance in complex samples. The more abundant proteins in the sample are preferentially and repeatedly detected by mass spectrometry, while the less abundant proteins are not detected because the peptide signal is too weak and is overwhelmed. Therefore, if the target protein to be detected in the sample is in low abundance, even if WB can detect it, mass spectrometry may not always detect it or may only detect a small number of peptides.

A: This is a normal phenomenon, the upstream and downstream are not one-to-one correspondence, the correlation coefficient between mRNA and protein is only 0.4~0.5. The expression of a protein is controlled by many factors, such as transcription factors, enhancers, repressors, DNA and RNA modifications, etc. in addition to the corresponding mRNA of the protein.

A: To evaluate the quality of proteomics data, one can examine various parameters such as the number of identified peptides and proteins, the level of protein coverage, the reproducibility of the results, and the accuracy of the quantification. Additionally, it is important to perform statistical analysis to identify significant differences between experimental groups and to verify the results using orthogonal methods such as Western blotting.

A: In principle, the more biological replicates the better, to exclude individual differences, the more accurate the screened differential proteins, and the higher the validation success rate. Considering the cost, the need for statistical analysis and the possibility of questioning by subsequent editors, it is recommended to have more than ten replicates per group for clinical samples and at least three replicates per group for samples from other sources.

A: There are several software packages commonly used for proteomics data analysis, including MaxQuant, Proteome Discoverer, Scaffold, and Skyline. These software packages are capable of performing tasks such as peptide identification, quantification, and statistical analysis.