We did an IP experiment and sent the results for mass spec. I confirmed by western blot (on the same sample) that some positive controls were present -- including the antigen. The mass spec results did not show these positive controls present in the results.
When we IP'd against an antibody for ProteinX, ProteinX was not present in the mass spec. Based on previous experiments we do know that ProteinX is indeed detectable by mass spec, and we also know based on Western, that it was present in our submitted sample. Any ideas what might have happened?
Not enough information provided to answer your question.
Mass spec's are NOT universal detectors. They do not and can not "see" everything.
For an LC/MS system to detect a compound, it must ionize well under the conditions used. This relies on many things, including, the concentration of the sample present, the experience and skill of the operator (This has the largest effect of all!), the type of MS system used, the mode the system is operated in (pos/neg) and of course, the many settings and parameters used to detect it (all of which can result in no signal, noise, signal or just about anything else).
For now, perhaps forget about the MS aspect and ask yourself if you can detect the sample using a good quality HPLC method with UV detection? Alternatively, if enough sample is available, try flow injection with the MS set up in a wide scan (look at the TIC) to see if anything is there and then a narrow SIM mode to look for fragments or clusters of the protein at higher energies.
*Most importantly of all, get some help locally with this (not the web) as a student would not be expected to do this on their own. It takes many years to learn how to analyze samples by HPLC and/or LC/MS and your focus should be on having someone help make sure you are pursuing a correct approach to solve the problem. Let them help you insure the instrument(s) and method(s) used are applicable to your sample and problem.
Not enough information provided to answer your question.
Mass spec's are NOT universal detectors. They do not and can not "see" everything.
For an LC/MS system to detect a compound, it must ionize well under the conditions used. This relies on many things, including, the concentration of the sample present, the experience and skill of the operator (This has the largest effect of all!), the type of MS system used, the mode the system is operated in (pos/neg) and of course, the many settings and parameters used to detect it (all of which can result in no signal, noise, signal or just about anything else).
For now, perhaps forget about the MS aspect and ask yourself if you can detect the sample using a good quality HPLC method with UV detection? Alternatively, if enough sample is available, try flow injection with the MS set up in a wide scan (look at the TIC) to see if anything is there and then a narrow SIM mode to look for fragments or clusters of the protein at higher energies.
*Most importantly of all, get some help locally with this (not the web) as a student would not be expected to do this on their own. It takes many years to learn how to analyze samples by HPLC and/or LC/MS and your focus should be on having someone help make sure you are pursuing a correct approach to solve the problem. Let them help you insure the instrument(s) and method(s) used are applicable to your sample and problem.
Protein is the macromolecule of a collection of amino acids.
If analyzed using MS, Proteins will certainly be fragmented into much smaller molecules (amino acids), you can learn from the type of fragmentation, and the molecular weight of the amino acids has a high abundance,
Can you provide the details of your experiment? Have you done desalting before running on the MS? Sometimes salts interfere with the ionization. Also, instrument used during the experiment is very important. Proper ionization voltage is the key to generate signal on the MS. Please tell us more.
More information is needed to answer this question.
1.) In top-down proteomics, where the intact protein is identified, usually only the most abundant proteins with molecular weights below 30-40kDa or detected. Thus, much of the proteome is below the detection limit.
2)In bottom-up proteomics, where proteins are digested into peptides prior to analysis, a peptide unique to the protein of interest is required. Not only must it be unique in the proteome, but it also must be in the detectable size range and with ability to ionize well.
3) Top-down in combination with IP can improve sensitivity.
4) In bottom-up, SRM/MRM assays can improve sensitivity.
5) Also not unusual for antibodies to have cross-reactivity. Therefore, protein X might in fact not be in the sample but the antibody cross-reacted with protein Y.
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