What causes synthesized \peptides to be over or under the expected weight when post-cleavage mass spectral analysis is performed? What causes multiple peaks for one peptide, by RP-HPLC?
Overweight: Most often reflects failure to remove all the side-chain protecting groups from all amino acids ("incomplete cleavage"). If insufficient amounts of scavengers were used, these protecting groups may reattach, OR permanently attach to another amino acid. If this occurs, it may be necessary to modify the system used for cleavage. Increased time, better mixing, or increased scavengers may be needed. This may be best determined by trial and error.
Underweight: Usually reflects a problem during the synthesis. If the run was done with conductivity or UV monitoring during Fmoc removal, it may be possible to modify the subsequent synthesis to avoid problems at specific areas of the sequence. An analysis of the probable secondary structure of the peptide chain may also be helpful for future strategies.
How can peptide synthesis amino acid cartridges leak and spill solvents during their activation and transfer to the RV?
If these cartridges are being reused, the NMP can cause them to swell, and they no longer fit or slide well in the guideway. If the guideway or the exterior of the needles have became dirty, this can also lead to misalignment. And if you forgot to remove the metal cap, the needle cannot penetrate the septum - this may cause a spill OR stop the run.
How often should the sound of the vacuum assist be heard on the 433A?
At most, once or twice a day. If more frequent, there may be a gas leak. Nitrogen pressure is used to generate the vacuum, which assists the opening of the valves. If there is no apparent gas leak, then it is possible that a valve has failed and the solvent leakage has damaged the vacuum ballast. Both the vacuum system and the valve block need inspection.
Why doesn't the 433A manual or the "quick start card" mention the need for an extra AA cartridge in the guideway at the start of a sequence?
The newest 433A User's Manual does cover this. The barcode reader is one position ahead (left) of the needle position. The extra (empty) is needed to prevent advancement of the first cartridge until after it is read.
Why is the conductivity so high in my peptide synthesis (monitored during deprotection)?
The meter detects any ionic species. A common cause of higher than expected values is a leak of a small amount of resin from the RV into the lines and up to the in-line filters. The use of old or poor quality piperidine or NMP may also give a high background. Standard conductivity measured in micro Siemens/cm is much higher than the sensitivity of this cell. A very small amount of ionic material caused a large change in the reading. Occasionally, Fmoc amino acids have ionic contaminants which give high readings. In-line filters may also be contaminated.
