In the August 1990 issue of ABRF NEWS, Bill Lane reported reusing pre-cycled filters for tryptic peptides. At the UWBC, we have also reused pre-cycled filters to sequence peptides. In one particular case we adopted this approach to perform some as-fast-as-possible sequencing for a user with a grant deadline. Normally we have 10 to 20 pre-cycled filters stockpiled ahead for rush times since we have found that filters can be stored with no adverse effects. At the time of the request for the emergency sequencing, we had only a single filter. These peptides all ended with the amino acids R-Famide, and were 7 or 8 amino acids long. In the first run with 55 pmols of peptide, we obtained 7 pmol of the final PTH-F residue with a PTH-(F/R) ratio of 0.36. Initial sequencing of the next peptide with 113 pmols yielded only 1 pmol of PTH-F with a PTH(F/R) ratio of only 0.06. This was after only 20 cycles on the filter. In the remaining runs, the most PTH-F we ever detected was 1.0 pmol and in some runs no PTH-F signal was obtained. The final peptide was sequenced with 92 cycles already run on the filter. This peptide turned out to be identical to the first peptide. Starting with 186 pmols--more than 3 times the material used in the first run--the yield at residue 4 was equal in the two runs and on the over-used filter, fell below that of the first run in subsequent cycles. While after 92 cycles on a filter the more rapid fall in per-cycle yield is not surprising, we were somewhat surprised that after only 20 cycles the final residue, PTH-F, was significantly reduced. Later we resequenced the same peptide on an unused pre-cycled filter and found that the PTH-F was easy to detect. The PTH-(F/R) ratio was essentially the same as the first run: 0.33.
We suggest that the decision to reuse a filter should be made with the final residues kept in mind. Tryptic peptides offer the advantage of ending either with an R or K which will be well retained by the filter. With F as the final residue, even with a preceding R, we found the limits to reusing filters to be more stringent than we expected. For neuropeptides or other peptides ending with hydrophobic residues, we prefer not to reuse filters.
RESPONSE FROM WILLIAM LANE
It should be emphasized that the original motivation for the technique was not throughput, (a benefit nonetheless), but rather improved interpretability of cycles 1 and 2 with samples of less than 10 picomoles. I agree with Cynthia and Ron that one should exercise caution when using this technique to analyze peptides with hydrophobic and/or uncharged C-terminal residues. As stated, our facility's most commonly analyzed peptides are from trypsin cleavages. Interestingly, a review of our data from 5 random chymotryptic peptides applied after > 40 cycles use, (7 - 58 picomoles, 7 - 16 amino acids long), gave final residue C-terminal ratios of 0.31 - 0.22. Each sequence analysis was fully readable and ended in the hydrophobic residue F, Y or L. Clearly a more formal study would be worthwhile.
Editor 's Note: Both of the above laboratories used ABI Model 477A instruments under similar conditions. The divergent observations from the two facilities suggest that the C-terminal amides in the UWBC peptides may be primarily responsible for the substantial different sequence performances. Other members with related data on multiple use of sequence filters are encouraged to share their experiences.
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Created: 15th August 1995
Last modified: 15th August 1995