FindMod is a tool that can predict potential protein post-translational modifications (PTM) and find potential single amino acid substitutions in peptides

3. Definition
is a tool that can predict potential
protein post-translational modifications (PTM)
and find potential single amino acid
substitutions in peptides.

4. de novo discovery of protein post-translational modifications (PTM).
It examines peptide mass fingerprinting results of known proteins for the
presence of 22 types of PTMs of discrete mass:
acetylation, amidation, biotin, C-mannosylation, deamidation, N-acyl
diglyceride cysteine (tripalmitate), FAD, farnesylation, formylation,
geranyl-geranyl, gamma-carboxyglutamic acid, O-GlcNAc,
hydroxylation, lipoyl, methylation, myristoylation, palmitoylation,
phosphorylation, pyridoxal phosphate, pyrrolidone carboxylic acid,
sulfatation.

5. The experimentally measured peptide masses are compared
with the theoretical peptides calculated from a specified
Swiss-Prot/TrEMBL entry or from a user-entered
sequence, and mass differences are used to better
characterise the protein of interest.
If a mass difference corresponds to a known PTM not already
annotated in UniProtKB/Swiss-Prot, "intelligent" rules are
applied that examine the sequence of the peptide of interest
and make predictions as to what amino acid in the peptide is
likely to carry the modification.

6. Protein Sequence to be characterize
Peptide Massses

7. 1.Protein Sequence to be characterized
You should specify the sequence of the
protein you would like to characterize and for
which you have determined a set of peptide
masses.

8. • If the Protein sequence is known
• If the Protein Sequence is not known

9. Enter the UniProtKB/Swiss-Prot ID code (e.g.
TKN1_HUMAN)
Or the protein accession number (e.g.
P20366).

10. you can enter the sequence of your protein of
interest, in single letter amino acid code, in
either upper or lower case.
The user is required to specify the biological
source of the query protein

11. Note
The characters O and U are not considered and will give
an error message. However, the residue J will be
treated as either Ile or Leu, which have the same
average and monoisotopic masses. The characters
B, X, or are accepted, but no masses are computed
for peptides containing one or more of these
characters.

12. 2.Peptide Masses
Enter the experimentally measured peptide
masses generated from the unknown protein
in the « Enter a list of peptide masses...»text
field, and separate them by spaces, tabs or
new lines.

13. NOTE
You can copy a list of peptides from Excel or
other applications and paste them directly
into the text field.
Avoid using peptide masses known to be from
autodigestion of an enzyme (e.g. trypsin!), or
other artefactual peaks (e.g. matrix peaks).
Upload a .pkm, .dta or text file

14. Uploading Peptide Masses
Upload the file directly from your computer
(1) Click on the on the «Browse...»button
(2) Select the file containing the relevant peptide mass
data.
(3) Click on the «Open» button
The peptide masses will then be extracted
automatically from this file

15. Expasy Tools
Go to Expasy site:
http://www.expasy.ch/tools/#proteome
Select FinMod Tool

16. FindMod View

17. Findmod View

18. Types of Supported Formats
(1) .pkm format
(2) Sequest format
(3) User Created Files

19. .pkm Format
.pkm format, produced by the Voyager software of
Perseptive Biosystems or the GRAMS software:
OP=0Center X Peak Y Left X Right X Time X Mass Difference NameSTD.Misc
Height Left Y Right Y %Height,Width,%Area,%Quan,H/A833.319 2189 833.260
833.378 0.016 0 0C0.? 0 762 762854.843 5078 854.769 854.917 0.001 0
0C0.? 0 3453 3453863.419 5108 863.064 863.775 0.001 0 0C0.? 0 3567
3567872.402 12519 872.347 872.456 0.002 0 0C0.? 0 11417 11417874.395
6730 874.331 874.460 0.002 0 0C0.? 0 3559 3559887.786 5903 887.540
888.031 0.003 0 0C0.? 0 4131 4131898.475 3329 898.416 898.534 0.006 0
0C0.? 0 1377 1377904.366 7432 904.199 904.533 0.001 0 0C0.? 0 5596
5596955.300 2598 955.229 955.371 0.011 0 0C0.? 0 1089 1089973.845 16689
973.749 973.941 0.001 0 0

20. 1.001833.319 2189844.333 0.0854.843
5078863.419 5108872.402 12519874.395
6730887.786 5903898.475 3329899.555
0.0904.366 7432955.300 2598973.845 16689
The first line is considered as a comment and is
ignored.

21. User Created Files
Any user-created files
can be uploaded if they correspond to the following rules:
The first line does not contain any mass value (if it does, this mass
value is ignored).
Lines containing masses must start with the mass, and the first 20
characters must not contain any uppercase letters.

22. NOTE
The upload option only works if you see a 'browse' button
next to the text entry field. This should be the case for most
recent web browser versions, e.g. Netscape 3.0 or higher, MS
Internet Explorer 4.0 or higher.

23. The user can specify whether the program should detect only
potential PTMs, only single amino acid substitutions or both.
The user can choose whether all peptide masses or only
those that have not been attributed a theoretical peptide in
this process should be examined for potential PTMs resp.
single amino acid substitutions.

24. ION MODE (MASSES AS [M] OR [M+H]+)
You can enter the masses of your peptides as [M] or as
[M+H]+, however you must select the appropriate button. If
you select the [M+H]+ button, all peptide masses calculated
from the database will have one proton (mass of 1 unit)
added before matching with user-specified peptides.

25. MASS TOLERANCE
Peptide masses can be specified in ppm (parts per million) or
in Dalton.

26. DIGESTION AGENT (ENZYME)
Specify the enzyme or chemical reagent that you used to
generate your peptides (see the corresponding section in the
PeptideMass instructions for the available enzymes and their
cleavage rules).

27. MISSED CLEAVAGES
In order to take into account partial cleavages, you can specify
a maximum number (0, 1, 2 or 3) of missed cleavage sites to
be allowed. If the maximum number of missed cleavages
entered is 1, all concatenations of two adjoining peptides are
also added to the list of theoretical peptides under
consideration.

28. SORTING OF PEPTIDES IN THE RESULT TABLES
Here you can choose if you would like the peptides to be
sorted by their mass (from smallest to largest) or by their
chronological order in the protein.

29. SEND THE RESULT BY E-MAIL
 Tick the « Send the result by e-mail » box. In the « Your e-mail:» text field
you should enter the correct e-mail address (e.g. name@unknown.ch) to
where the results should be sent.
 The email option is recommended, in particular for queries with a high
number of peptide masses. This avoids timeouts («document contains no
data») which can occur for the on-line option: the browser interrupts the
connection with the program if the search is not terminated after a certain
time (usually about 3 minutes).
 Note that email results are sent in form of a html file, in exactly the same
format as on-line, and that there is no loss of functionality compared to
on-line display.

30. RESET AND PERFORM BUTTONS
Once you have filled in the form according to your needs, press the button
"Start FindMod". If you have made a mistake and would like all fields to
be reset to their default values, press the Reset button.

31. OUTPUT FORMAT
o Header
o up to three tables.

32. HEADER
The header contains information about the submitted
protein:
 A link to the UniProtKB/Swiss-Prot or UniProtKB/TrEMBL entry
 The description line (if the protein is in UniProtKB), pI and
molecular weight.

33. Then the input parameters are listed, followed by an active
link to PeptideMass. This allows the user to perform a
theoretical cleavage of the protein of interest.

34. TABLES
The tables report the peptides whose experimental masses
match unmodified or modified theoretical digest products of
the protein of interest:
 The first table reports matches to theoretical digest products as
unmodified, modified with the annotations in UniProtKB/Swiss-Prot and
chemically modified as specified in the input form.
 The second table reports those user masses which differ from a
theoretical database mass by a mass value corresponding to one of the
considered PTMs.
 The third table shows potential single AA substitutions detected by mass
difference.

35. FINDMOD OUTPUT
}
unmodified peptides,
modified peptides
known in SWISS-PROT
and chemically modified
peptides

36. putatively modified
peptides predicted
by mass differences
+ putative AA substitutions
{

37. -potentially modified peptides that agree with rules are listed
- amino acids that potentially carry modifications are shown
- peptides potentially modified only by mass difference
- predictions can be tested by MS-MS peptide
fragmentation

38. At the end of the output page the user will find a list of those
entered matches which did not match in any of the above
tables (if any).