In instances where the peptide sequence has ended and the sequence chromatogram has returned to baseline, we routinely reuse glass filters without reapplying polybrene, when sequencing small peptides from proteolytic digestions. From informal discussions at the MPSA meeting in Sweden, it is clear that many other laboratories also follow this practice, while others are unaware that this approach is feasible.
As a rule, we use a filter continuously for 40 - 60 total cycles, e.g. 2 - 4 average length tryptic peptides. Generally 4 - 5 cycles after the carboxyl-terminal is reached, the sequence chromatogram is back to baseline and the next sample is loaded directly and immediately sequenced. In our extensive experience over the last several years, we performed about 60 sequences/ month on 2 sequencers dedicated to peptide work and observed no adverse effects of running a series of peptides on a single filter in the low picomole range. In fact there are two distinct advantages to this practice: increased throughput by avoiding the 2 - 3 hour precycling required when polybrene is reapplied, and decreased noise due to polybrene artifacts in subsequent sequences. The latter advantage is most important to us and we reserve our most sensitive samples [ < 10 picomoles] for the second sequence in a series to improve interpretation of the earlier cycles.
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Created: 10th August 1995
Last modified: 10th August 1995