Hi,
I am trying to develop an assay to detect a conjugated peptide in plasma. I am essentially brand new to MS but have LC experience. The analyte of interest is a bisphosphonate tethered to parathyroid hormone (1-34) with a MW of 5868 (BP-PTH). Currently I am doing direct injections (no column) into a trip quad MS. I am able to detect the z=5, z=6, z=7 charged species of the conjugated peptide consistently.
However the problem arises when I use TMS-DAM to derivative the peptide by neutralizing the phosphate groups via methylation. We have discovered that the negatively charge bisphosphonate moieties actually bind to the metal tubing and metal columns in LC, which prevents accurate quantitation. Therefore, derivatization is required. I am able to get consistent detection and retention times (indicating no column binding) of the TMS-DAM derivatized BP-PTH in HPLC. When I use the derivatized BP-PTH with the phosphates methylated, I get much lower sensitivity in mass spec and I cannot get consistent precursor ions in Q1 scans. I get similar fragmentation each time, but it seems every new run the fragments have m/z values that differ in a range of +/- 15 m/z. The fragments are always similar in mass, but I can never get the exact same fragments to select precursor ions. The only consistent precursor ions I get is an intense peak at 306 m/z and 229 m/z. These are the only consistent peaks. Has anyone encountered this before? Why do I get all these issues once I derivatize?
Please look at the attached powerpoint. In the meantime I am trying to get connected with some LC-MS/MS experts at my university.
Noel W Davies This was my original thought- that the methylation is inconsistent. I believe that TMS-DAM can also methylate alcohols, so I figure that the serine residues could also be methylated. Taking into account 4 possible methylations on the BP group and then 6 possible methylations on the peptide portion, in theory the largest MW after derivitzation with full methylation would be 6012.56 Da. I will look into some of the spectras I recorded the other day and see if I can identify any of these peaks with correct corresponding MW.
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