Sequencing yields from in gel digests
Kenneth Williams (Kenneth.Williams@yale.edu)
Thu, 05 Mar 1998 17:14:48 -0500
In regard to the recovery of peptides from in gel digests, there are a few
large scale studies that provide relevant data. In the 1997 ABRF Study
carried out by the Internal Protein Sequencing Group, hydrolysis and ion
exchange amino acid analysis (of selected gel bands) indicated that each of
the bands that were distributed contained an average of 52 pmol protein.
In comparison, the median sequencing yield for Phe (3) (obtained by 16
participating laboratories that identified and sequenced the "target"
peptide from in gel digests) was 8.9 pmol or 17%. Previously, we reported
(Williams et al, Techniques VIII (79-90)) an average initial sequencing
yield of 12% from 409 peptides obtained from 191 "unknown", user-submitted
proteins subjected to in gel digestion. In this instance the amount of
protein was quantified by hydrolysis/amino acid analysis of an aliquot of
the Coomassie Blue stained gel band and the %initial sequencing yield
obtained by dividing the peptide sequencing yield by the (amino acid
analysis) estimated amount of protein digested in the gel band.
It is important to note also that with some proteins, losses may occur
during washing of stained gel bands prior to in gel digest (see the above
reference) and that additional losses may also occur prior to this point,
that is, during SDS PAGE, and staining and destaining of the gel.
This year's Internal Sequencing Study, which will be summarized at the ABRF
Meeting, will provide additional data relating to the recovery of peptides
from in gel digests - as well as lots of other interesting results.
Ken Williams