Transcript Facts and Fallacies

Facts and Fallacies
about de Novo Sequencing &
Database Search
1. There are a large number of
high quality spectra left
unassigned after DB search.
True
False
Unassigned Spectra in ABRF/iPRG 2011 Study
Unassigned Spectra
PEAKS DB
De novo sequencing
PEAKS PTM
SPIDER
•
•
•
•
Nonspecific trypsin cleavages
Novel peptide/incomplete database
PTM
Mutations
2. Nonspecific cleavage, PTM,
mutations and novel peptides
are the main reasons for the
unassigned spectra.
True
False
Average Software Misses Peptides
3. De novo sequencing is slow.
True
False
Speed
• PEAKS 6 de novo sequence 15 spec/second.
– Intel i7 Quad Core, 8GB RAM.
– Trypsin
– Orbitrap CID MS/MS, mostly charge +2/+3
• PEAKS 7 (coming soon):
– Improve speed on high charge states and longer
peptides.
– Add 8 core support in standard (desktop) license.
4. De novo should be done after
DB search.
True
False
DB search
DB peptides
Unassigned spectra
de novo seq.
de novo peptides
Order of de Novo and DB
• Better conduct de novo on all spectra.
– De novo not slow, and computing is cheap.
– De novo provides independent validation for DB result.
# consensus AA
(de novo vs. DB search)
false
without with
de novo de novo
true
true
score
5. My protein sequence is
confirmed with two unique
peptide hits.
True
False
Routine Full Protein Coverage
• For regular proteins, full sequence coverage
can be routinely achieved with
– 3 or more enzyme digests, and
– multiple algorithms in PEAKS 6.
• For highly variable proteins (such as
antibodies), BSI offers data analysis service for
antibody sequencing.
6. If a peptide is identified with
1% FDR, then it’s sequence is
99% correct.
True
False
Peptide Validation vs. Amino Acid
Validation
You are confident about the peptide sequence only if
• you can de novo sequence it, and
• the de novo sequence matches the database peptide.
7. I don’t need de novo
sequencing if I have a protein DB.
True
False
8. Target-decoy provides a
reliable result validation for
every DB search engine.
True
False
Target-Decoy Incompatible with Certain
Highly Optimized Search Engines
weak hits
confident protein
weak protein
• Adding “protein bonus” to peptide hits increases accuracy.
• But it creates bias between target and decoy.
– In extreme, bonus is so large that only peptides from target proteins
are selected.
– This gives the wrong impression that FDR=0, while there are still false
peptides in the result.
Decoy Fusion Is A More Powerful
Validation Method
weak hits
confident protein
weak protein
• Decoy fusion append a decoy sequence to each
protein.
• Recreates the balance.
• The built-in validation method since PEAKS 5.3.
9. Combining 1% FDR results of
multiple engines gives 1% FDR.
True
False
Error Accumulation
Target(decoy)
FDR%
PEAKS DB
3870(38)
1%
Mascot
2369(23)
1%
Correct < sum of the two
Error ≈ sum of the two
PEAKS DB
1696(37)
2.4%
Mascot
2174(1)
0.1%
195(22)
13%
Combined FDR = 1.5%
• In PEAKS, the inChorus algorithm automatically selects a less
than 1% common FDR for each engine so that the combined
FDR is approximately 1%.
10. There is no automated way
to validate de novo sequencing
results.
True
False