azim58 - my annotations from paper 4-16-13 dot 0909


Bioinformatic requirements for protein database searching using
predicted epitopes from disease-associated antibodies
Page: 1
Page: 1 Author: Kurt Whittemore Subject: The E-MAP process first involves
re- construction of a predicted epitope using a peptide
com- binatorial library. We then search the protein database for closely
matching amino acid sequences. Date: 2013-04-14 14:38:
51-07
Page: 1 Author: Kurt Whittemore Subject: 1) short predicted epitopes
yield too many irrelevant matches from a database search
and 2) the epitopes may not accurately represent the native antigen with
sufficient fidelity. Date: 2013-04-14 14:39:24-07
Page: 1 Author: Kurt Whittemore Subject: We find that epitopes generally
need to have at least seven amino acids, with an overall
accuracy of >70% to the native protein, in order to correctly identify
the protein in a nonredundant protein database search. Date:
2013-04-14 14:40:34-07
Page: 1 Author: Kurt Whittemore Subject: we demonstrate the efficacy of
paired epitope searches Date: 2013-04-14 14:40:52-07
Page: 1 Author: Kurt Whittemore Subject: It has not previously been pos-
sible, however, to use these epitope motifs to identify un-
known target proteins from the entire protein database. Date: 2013-04-14
14:45:33-07
Page: 1 Author: Kurt Whittemore Subject: A short motif (4–6 amino
acids) does not possess enough in- formation content to
uniquely identify a candidate antigen in broad bioinformatic searches.
The list of search results re- trieved from the nonredundant (nr)
database is usually large, with hundreds or thousands of hits effectively
burying the true matching protein in the noise of extraneous
results. Date: 2013-04-14 14:46:17-07
Page: 1 Author: Kurt Whittemore Subject: E-MAP is for identifying
proteins that are immunoreactive with an antibody without any
prior information as to the identity of that protein. Date: 2013-04-14
14:47:31-07
Page: 1 Author: Kurt Whittemore Subject: PSSM, position-specific scoring
matrix; Date: 2013-04-14 15:05:10-07
Page: 2
Page: 2 Author: Kurt Whittemore Subject: Sticky note Date: 2013-04-14
14:49:54-07
etiology the cause or origin of a disease
Page: 2 Author: Kurt Whittemore Subject: Phage libraries contained
rationally designed combinatorial librar- ies of peptide
sequences inserted into the N
terminus of the cpIII minor coat protein of the M13 bacteriophage. The
libraries were supplied by Dyax
Corp. (Cambridge, MA). The libraries termed TN6 and TN10 contained two
conserved cysteine residues separated re- spectively by
four or eight amino acids. The cysteines formed a disulfide bridge,
creating a conformationally constrained ring (10). Date:
2013-04-14 14:51:46-07
Page: 2 Author: Kurt Whittemore Subject: This negative depletion step
removes phage that may bind to constant regions of mouse
IgG. Date: 2013-04-14 14:52:57-07
Page: 2 Author: Kurt Whittemore Subject: Phage par- ticles that bound to
the mAb-coated beads were eluted with 0.1 mol/L
glycine-HCl (pH 2.2) containing 1 g/L bovine serum albumin Date:
2013-04-14 14:54:02-07
Page: 2 Author: Kurt Whittemore Subject: The variable regions of the
inserts were transcribed into the FASTA form and submitted
to MEME (Multiple EM for Motif Elicitation, available at meme.sdsc.edu/
meme). Date: 2013-04-14 14:58:51-07
Page: 2 Author: Kurt Whittemore Subject: To carry out bioinformatic
searches using a single motif, the PSSM was submitted to the
MAST (Motif-Alignment and Search Tool) utility (meme.sdsc.edu/meme), to
be searched against the nr protein data- base while
allowing a maximal E value (expectation value). The first 500 hits were
then screened for the presence of the known target. F Date:
2013-04-14 15:00:43-07
Page: 3
Page: 3 Author: Kurt Whittemore Subject: The E value can then be thought
to represent the expected number of sequences in a
random database of equal size that would match the motif(s) at least as
well. Date: 2013-04-14 15:03:17-07
Page: 3 Author: Kurt Whittemore Subject: short sequences of predefined
length N were se- lected randomly from the NCBI nr
protein sequence database. Date: 2013-04-14 19:10:28-07
Page: 3 Author: Kurt Whittemore Subject: These sequences were then used
to construct a position specific probability matrix, with
the degree of residue conservation at each position perturbed by a
Gaussian function around the average conservation,
C. Date: 2013-04-14 19:10:59-07
Page: 3 Author: Kurt Whittemore Subject: Sequence Generation Date:
2013-04-14 19:12:09-07
Page: 3 Author: Kurt Whittemore Subject: zoops model and user-defined
restriction of the motif length. Date: 2013-04-14 19:13:
05-07
Page: 3 Author: Kurt Whittemore Subject: For pairwise searches, we set
the MAST threshold expectation value (E value) to
Epitope-mediated Antigen Prediction
10 and the threshold value for motif display to p
0.0001. Date: 2013-04-14 19:15:11-07
Page: 3 Author: Kurt Whittemore Subject: Sticky note Date: 2013-04-14
19:17:15-07
Does this represent all amino acids?
Page: 3 Author: Kurt Whittemore Subject: MEME creates a consensus motif
profile, capturing each phage clone’s sequence infor-
mation in PSSM, a two-dimensional numeric array. Date: 2013-04-14
19:19:12-07
Page: 3 Author: Kurt Whittemore Subject: Using such a profile in a
bioinformatic search offers distinct advantages. Instead of
searching with a single “best-guess� query repre- senting the
dominant motif, the queried profile considers a larger number of
combinatorially weighted sequences, aver- aging around the dominant
motif. Date: 2013-04-14 19:19:51-07
Page: 4
Page: 4 Author: Kurt Whittemore Subject: The combined statistical power
of a pairwise search, however, is sufficient to narrow the
list to a small number of antigen candidates. We use the MAST (Motif
Align- ment and Search Tool) utility (16, 17) to perform single
and pairwise motif searches against the nr protein database. Date:
2013-04-14 19:21:28-07
Page: 4 Author: Kurt Whittemore Subject: There are two important
variables for identifying proteins from only short linear epitopes:
1) the length of the epitope and 2) the fidelity with which the predicted
epitope matches the actual sequence in the protein database
(average motif conservation). Date: 2013-04-14 22:06:32-07
Page: 4 Author: Kurt Whittemore Subject: For the process to yield
accurate da- tabase matches, how long and accurate must the
predicted epitope be? Date: 2013-04-14 22:06:54-07
Page: 5
Page: 5 Author: kwhittem Subject: Highlight Date: 2013-04-15 10:26:32-07