IP-MS Sample Preparation Q&A

A: If detecting a single protein band, cut only the position of that protein band. If detecting multiple protein bands, cut each band separately and then combine them. If detecting the entire lane of proteins, it is recommended to cut the gel or separation gel after running for a short time to concentrate the proteins.

A: For a single protein band, the protein quantity should be no less than 20μg. For multiple protein bands, the protein quantity for each band should be no less than 20μg. For the entire lane of proteins, the total protein quantity should be no less than 50μg (with lower-abundance proteins less easily detectable). For purified protein interaction bands, it is recommended that the protein quantity before purification be no less than 5mg (low protein quantity before purification may lead to lower interaction protein content after purification, resulting in fewer detected proteins). Protein quantity in gel strips should not be judged based on the color intensity of gel staining, as the longer the staining time, the darker the color.

A: No, when purifying proteins, antibodies are typically used (not required for affinity purification). If SDS-PAGE separation is not performed, antibodies may interfere. If studying the interacting proteins of the target protein, SDS-PAGE separation is necessary to exclude the target protein (which is generally highly expressed, and not excluding it may cause detection interference).

A: Do not use glutaraldehyde in silver staining reagents, as it modifies proteins and may affect subsequent protein identification.

A: Gel strip identification is a qualitative analysis, not a quantitative one. If you need to know the abundance of a protein in a sample, you can make a rough estimate based on the number of spectra for each protein. The more spectra, the higher the abundance of that protein. If comparing the abundance of the same protein in multiple samples, iTRAQ or label-free methods can be used for relative quantification.

A: In gel strip identification, the standard for identifying proteins is having at least one unique peptide. The listed proteins are those identified, along with their corresponding scores. You can filter target proteins based on their scores, combined with molecular weight, isoelectric point, and other information.

A: Protein mass spectrometry identification involves merging peptides into proteins. Therefore, some identified proteins may be homologous proteins of the target protein or subunits of that protein. Artificial or natural modifications and degradation of proteins can also affect the position of the protein on the gel. Generally, the higher the Mascot identification score, the higher the reliability. Additionally, factors such as molecular weight, isoelectric point, and peptide coverage can be considered to filter for highly reliable proteins.

A: Firstly, for the ion score of peptide segments and spectrum matching (Ion Score), Mascot provides an identification threshold (Identity Score). Peptide segment matches with an ion score greater than the identification threshold are considered reliable, while matches below the threshold are considered unreliable and filtered out. For different ion scores, Mascot determines the threshold based on the number and score of candidate peptide segments, so the threshold is not a fixed value. To ensure the credibility of proteins, the number of unique peptides specific to a protein (UniQue Peptides) can be considered. Only when a protein has at least 1 unique peptide is it considered identified, and a score is provided.

A: The proteome refers to all the proteins expressed by a species, cell, or tissue. Proteome-wide analysis aims to identify as many components of peptide and protein mixtures in a sample as possible. Mass spectrometry-based comprehensive analysis provides reference information for high-throughput protein quantification and modification analysis. High-precision liquid chromatography-mass spectrometry technology for proteome-wide analysis allows the analysis of protein types in complex samples. It also enables various bioinformatics analyses, including protein identification, GO classification, and pathway analysis, providing powerful tools for proteomics.

A: Any species with sufficient data in the proteome database, a known genome, or abundant EST (or transcriptome) data can undergo proteome-wide analysis. For species that do not meet these conditions, simultaneous genome or transcriptome sequencing can support proteome-wide analysis. Therefore, theoretically, all species can undergo proteome-wide analysis.

A: 1) SDS-PAGE-LC-MS/MS analysis separates proteins in the sample using electrophoresis, achieving good separation and high resolution for proteins with molecular weights between 10 kDa and 100 kDa. However, it faces challenges in separating proteins with molecular weights less than 10 kDa or greater than 100 kDa, low-abundance proteins, extremely acidic or basic proteins, and hydrophobic proteins (such as membrane proteins). 2) HPLC-LC-MS/MS analysis separates proteins in the sample using HPLC, offering high separation efficiency, sensitivity, wide applicability, and fast analysis. This method is superior to SDS-PAGE in separating acidic and hydrophobic proteins. However, HPLC has the drawback of the "extra-column effect," where any diffusion and retention of separated substances significantly broaden chromatographic peaks, leading to false positives.

A: If the analysis targets include highly hydrophobic proteins like intrinsic membrane proteins and proteins with molecular weights greater than 100 kDa or less than 10 kDa, it is recommended to use the HPLC-LC-MS/MS analysis method. If the analysis targets are not predominantly hydrophobic proteins like membrane proteins and the molecular weights are concentrated in the range of 10–100 kDa, the SDS-PAGE-LC-MS/MS analysis method is suggested.

A: This depends on the complexity of the analyzed sample, the completeness of the database, protein content, and the degree of separation of the protein composition. Typically, comprehensive analysis can identify 1000–3000 types of proteins.

A: Three common modifications: oxidation, alkylation, acetylation.

For phosphorylation and ubiquitination, enrichment is usually required. In their natural state, the levels of phosphorylation and acetylation are very low. Tandem mass spectrometry selects top N based on the abundance of parent ions for secondary fragmentation. Without prior enrichment, the identified phosphorylation and acetylation peptides may be limited.

The peptide information table includes details about which amino acid site underwent the modification.

A: Yes, it is necessary. Adding a protease inhibitor prevents protein degradation, and adding a phosphatase inhibitor prevents the loss of phosphorylation.

A: Both methods are acceptable. Generally, with fewer products, silver staining has higher sensitivity and is recommended. Coomassie staining has lower sensitivity, and bands may not appear, but proteins can still be identified.