Do you have an antibody raised against your peptide? If so, you could do a simple dot blot to detect it - though this obviously wouldnt work if your peptide is cleavage fragment from your large protein... Also if you have the antibody, then you could possibly try SELDI-TOF-MS. If not, maybe MALDI. I'm no expert on either of these techniques, but i believe they can be used for quantitative analyses with the right controls.

Thank you Victoria, do not have an antibody to the peptide, and there will be several, so I need to be able to find a way to quantitate them at the beginning and the end of the assay.

Does the protein and enzyme will interact ? What is the sequence of the peptide. Maybe if the aromatic residues are present you should follow them. Alternatively labeling of both will help. But you should provide more details.

Should be protein and peptide. Sorry

If you would like to monitor and quantitate small peptide and larger protein separately, centrifugal filtration following any sensitive protein estimation (fluorescence) may be done. However, MRM also called as SRM analysis after labeling with ITRQ is the method of choice for accurate quantification. One can use LC-QTOF-MS/MS of LC-QTRAP-MS/MS equipments.

The peptide "may be" a substrate for the enzyme, so I want to monitor the peptide over time to see if it is being degraded or not.

the SPR will help in sense of binding. If the peptide get digested no binding. Capillary electrophoresis is non denaturing alternative for such experiment. Or labeled the peptide by one or two fluorophores and use fluorescence to find out. NO other clue comes to my mind.

Something simpler -- and very powerful, especially, when it comes to the possibility of degradation: record the radius of gyration over time. Light scattering (either dynamic or static, doesn't matter) would suffice, provided that you operate with the dilute solution of proteins.

If I got your question right, this is the answer, otherwise, feel free to ask further.

Good luck!

Artem

For such a small peptide and its degradation product, mass spectrometry is the method of choice. Not only will you get their amounts, but you can detect any differences in cleavage products based on differences in LC retention times and MS spectra.

Maybe 1H NMR can be of help, but its suitability may depend on the specifics of your system...

If you can work at about 100uM peptide concentration (in about 650uL) with some tricks you could monitor your system inside the NMR tube without any perturbation of your system. With some controls you can establish if a standard molecule used for chemical shift referencing (DSS or TSP) can be used as an internal reference for concentration determination (using a concentric tube with D2O and DSS may be even better)

You only need about 550uL in for NMR so you can collect samples over time for SDS analysis of the 65kDa protein). For quantifying the peptide, If the concentration of the 65KDa protein is much smaller than that of the peptide you will have no problem. If it is high (0.2:1 and above) you may need to use a sequence that filters out bigger proteins (1D CPMG with about 15-30ms delay)

Alternatively you can collect a spectrum of your peptide prior to the addition of the larger protein and then after some incubation, separate the two with a 10kDa MWCO filtering device and collect a spectrum of the flow-through. In this case I would do the reaction in the presence of a third molecule (DSS or TSP) that will be used for quantification and I would also check if these standards interact with any of your peptides and or the filter)