What is the relationship between PRM technology and the Shotgun method commonly used in proteomics?
Answer: Shot-gun and PRM are two different mass spectrometry scanning methods. Shotgun is a large-scale scanning method where the goal is to detect as much protein as possible in the sample. It usually has a bias toward easy detection of higher-abundance proteins. PRM, on the other hand, is a scanning method for specific proteins of interest, with the goal of achieving precise quantification of the target protein.
When would a technique like PRM be used?
Answer: PRM allows selective detection of target proteins, target peptides (e.g. peptides undergoing post-translational modifications), thus achieving target validation and relative or absolute quantification of the target protein/peptide. It is used for validation experiments after broad screening.
WB can be used for target protein quantification, why choose PRM?
Answer: The quantification of target proteins can indeed be achieved by both methods.
WB is an antibody-dependent assay, and its effect is closely related to the quality of the antibody and the experimental operation; WB is a semi-quantitative analysis based on the brightness of the chromogenic bands. The quality of antibodies currently on the market varies. There are no commercial antibodies available for many proteins. The cycle time for preparing monoclonal antibodies is long. These factors limit the effectiveness of WB.
PRM, on the other hand, is an antibody-independent, mass spectrometry-based targeted quantification technique. PRM is highly sensitive and specific, with a quantification range of more than 4 orders of magnitude, enabling precise quantification of proteins in the true sense. In addition, the throughput of PRM is higher. Up to dozens of proteins can be quantified simultaneously in one PRM experiment, which is also difficult to be achieved by WB.
What can PRM be used for?
A: Experiments related to protein or peptide quantification in research work can be achieved by PRM technology. For example, to verify the changes of certain proteins in quantitative proteomics; relative and absolute quantification of peptides; want to do protein quantification but no antibody; quantification for protein modification sites; want to quantify multiple proteins at the same time, etc., all can be done by PRM.
Is technical duplication mandatory for PRM validation of biological samples?
A: Biological replicates are used for PRM validation.
What is the advantage of MRM, PRM for proteomics to observe the differential proteins between groups and also for relative quantification of proteins?
A: Targeted MRM, PRM quantification is more accurate and also enables absolute quantification of proteins.
Can PRM be done without a standard curve?
A: The standard curve is only used for absolute quantification, and is not needed for relative quantification.
How many proteins of interest in the proteomic results are normally selected for PRM validation?
A: There is a certain chance that the proteomic results cannot be validated by PRM, or the validation results are consistent, so it is recommended to have at least 5 proteins.
Can PRM be used for low abundance proteins like transcription factors? Is it not suitable to use if the protein is not detected by proteomics?
A: If the proteome has been graded in depth and the protein is still not found, then it is very likely that PRM will not detect the protein either.
With the transcriptome data, is it possible to find the corresponding proteins from the differential genes for PRM validation? Is it highly feasible?
A: Yes, it is possible to try, but whether PRM can be detected depends on the abundance of the gene after translation into protein and the consistency of expression at transcriptional level and protein level. Ideally, it is better to have proteomic results followed by PRM validation.
Does phosphorylated proteomics mainly analyze changes in phosphorylation modification sites and modification levels, but not protein abundance? Do I need to do PRM to verify protein abundance?
A: Phosphorylated proteins cannot be analyzed for protein abundance because the peptides have been enriched and will filter out many unmodified peptides.
The need for PRM validation is determined by the purpose of the study. If you want to verify that the phenotype is primarily due to phosphorylation of a site rather than protein expression levels, PRM is needed to verify the expression abundance of the unmodified peptide corresponding to the phosphorylated site. This will accurately reflect whether the site is a change in the level of modification or a change in the overall level of the protein.
Is there any requirement for protein to perform PRM after iTRAQ has been done?
A: The mass spectrometry data of the protein needs to be evaluated by engineers in advance to assess the feasibility of the detection. From experience, some of the data will be less reproducible because iTRAQ is generally graded and the quantitation principle is quite different from PRM. We can provide some suggestions for protein selection for PRM based on the iTRAQ results.
Do I need to do PRM validation after I have done TMT quantification?
A: Yes, the later validation is increasingly needed at present.
Do internal standard peptides have to be isotopic peptides? Can specific unlabeled peptides added exogenously be used?
A: Yes, for example, galactosidase is often used as an internal reference for some samples, and this enzyme is only expressed in microorganisms. However, it can only be used as an internal reference for normalization, not for standard curve.
Is it generally recommended to use the same protein sample or different extracted protein samples for validation?
A: It depends on the bias of the experimental purpose. Identical samples have a higher consistency match of results and are used to validate experimental results. Different batches of samples are more scientifically sound and are used to validate biological conclusions, but there is a greater risk of inconsistency.
When proteins differ in proteomics, will there be no difference or opposite expression after PRM validation?
A: This is possible. The large-scale screening of untargeted proteins has a certain false positive rate and the quantitative results are not as accurate as PRM.
How can we be sure that a peptide will not have a missed cut site?
A: It can only be determined from the pre-experimental results, not from the theoretical sequence.
