De Novo Sequencing v.s.
Database Search
Bin Ma
School of Computer Science
University of Waterloo
Ontario, Canada
Two Computational Approaches
MS/MS Spectra
1
2
de novo
sequencing
database
search
peptides
peptides
protein DB
Is This a True Peptide Identification?
Take Advantage of Both Approaches
All Spectra
DB search
No
De Novo
Assigned?
Yes
novel peptides
DB peptides
Facts and Fallacies
1. De novo is harder.
• True. It can be regarded as a “universal” DB search.
2. De novo is slow.
• False. PEAKS processes 15 spectra/sec on a PC.
3. De novo is less reliable.
• Depends.
Goals of This Talk
• A “fair” comparison
• A “real” combination
What Is a Fair Comparison?
• The ability to report a long sequence tag
– long enough to uniquely identify the correct peptide from
the same database.
Experiment 1.
• Purpose: to check the length of correct de
novo tags on good quality spectra.
• Trypsin digest of Human tumour cell; LTQOrbitrap CID.
• Mascot DB search; 1% FDR.
• Compare PEAKS de novo sequences with DB
search results.
percatage in Mascot high confidedent PSM
Distribution of Correct Tag Length
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
≥5
≥6
≥7
≥8
≥9
# of correct amino acid found by PEAKS de novo sequencing
≥10
“De Novo Only” Peptides
Experiment 1 Conclusion.
• De novo sequencing performs well in terms of
finding long sequence tags.
• De novo also finds “de novo only” peptides.
Experiment 2.
• Purpose: To test de novo tags’ ability for identifying the
peptides from the database?
• For each de novo sequence, reports the “best-matching”
peptide from the database.
• Use target-decoy to determine the FDR.
Experiment 2 Conclusion
• De novo tags have better and complementary
ability in selecting the correct peptides from
DB.
• This might be due to the choice of the
software.
Experiment 3. De Novo Assisted DB Search
# matched amino acids
between de novo & DB search
best
separation line
x+4y
Mascot Score
PEAKS DB
Mascot
PEAKS DB
2.5%
FDR
2.0%
1.5%
1.0%
0.5%
0.0%
0
500
1000
1500
2000
2500
# of PSM
3000
3500
4000
Conclusion
MS/MS Spectra
De novo both helps to
improve DB search, and
reports novel peptides.
De Novo
improves
no
DB search
Found?
yes
De novo only
peptides
Improved DB
search results
This is the default
workflow of PEAKS 5.3.