Definition          is a tool that can predict potentialprotein post-translational modifications (PTM)and find potential s...
de novo discovery of protein post-translational modifications (PTM).It examines peptide mass fingerprinting results of kno...
The experimentally measured peptide masses are comparedwith the theoretical peptides calculated from a specifiedSwiss-Prot...
Protein Sequence to be characterizePeptide Massses
1.Protein Sequence to be characterizedYou should specify the sequence of theprotein you would like to characterize and for...
• If the Protein sequence is known• If the Protein Sequence is not known
Enter the UniProtKB/Swiss-Prot ID code (e.g.TKN1_HUMAN)Or the protein accession number (e.g.P20366).
you can enter the sequence of your protein ofinterest, in single letter amino acid code, ineither upper or lower case.The ...
NoteThe characters O and U are not considered and will give  an error message. However, the residue J will be  treated as ...
2.Peptide MassesEnter the experimentally measured peptidemasses generated from the unknown proteinin the « Enter a list of...
NOTEYou can copy a list of peptides from Excel orother applications and paste them directlyinto the text field.Avoid using...
Uploading Peptide MassesUpload the file directly from your computer(1) Click on the on the «Browse...»button(2) Select the...
Expasy Tools         Go to Expasy site:         http://www.expasy.ch/tools/#proteomeSelect FinMod Tool
FindMod View
Findmod View
Types of Supported Formats(1) .pkm format(2) Sequest format(3) User Created Files
.pkm Format.pkm format, produced by the Voyager software ofPerseptive Biosystems or the GRAMS software:OP=0Center X Peak Y...
1.001833.319    2189844.333    0.0854.843 5078863.419 5108872.402 12519874.395 6730887.786 5903898.475 3329899.555 0.0904....
User Created FilesAny user-created files  can be uploaded if they correspond to the following rules:     The first line do...
NOTEThe upload option only works if you see a browse buttonnext to the text entry field. This should be the case for mostr...
The user can specify whether the program should detect onlypotential PTMs, only single amino acid substitutions or both. T...
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 t...
MASS TOLERANCEPeptide masses can be specified in ppm (parts per million) orin Dalton.
DIGESTION AGENT (ENZYME)Specify the enzyme or chemical reagent that you used togenerate your peptides (see the correspondi...
MISSED CLEAVAGESIn order to take into account partial cleavages, you can specifya maximum number (0, 1, 2 or 3) of missed ...
SORTING OF PEPTIDES IN THE RESULT TABLESHere you can choose if you would like the peptides to besorted by their mass (from...
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...
RESET AND PERFORM BUTTONSOnce you have filled in the form according to your needs, press the button"Start FindMod". If you...
OUTPUT FORMATo Headero up to three tables.
HEADER  The header contains information about the submitted  protein: A link to the UniProtKB/Swiss-Prot or UniProtKB/TrE...
Then the input parameters are listed, followed by an activelink to PeptideMass. This allows the user to perform atheoretic...
TABLES  The tables report the peptides whose experimental masses  match unmodified or modified theoretical digest products...
FINDMOD OUTPUT      }      unmodified peptides,      modified peptides      known in SWISS-PROT      and chemically modifi...
putatively modified peptides predicted by mass differences+ putative AA substitutions                              {
-potentially modified peptides that agree with rules are listed- amino acids that potentially carry modifications are show...
At the end of the output page the user will find a list of thoseentered matches which did not match in any of the abovetab...
FindMod
FindMod
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FindMod

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

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Transcript of "FindMod"

  1. 1. Definition is a tool that can predict potentialprotein post-translational modifications (PTM)and find potential single amino acidsubstitutions in peptides.
  2. 2. de novo discovery of protein post-translational modifications (PTM).It examines peptide mass fingerprinting results of known proteins for thepresence 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.
  3. 3. The experimentally measured peptide masses are comparedwith the theoretical peptides calculated from a specifiedSwiss-Prot/TrEMBL entry or from a user-enteredsequence, and mass differences are used to bettercharacterise the protein of interest.If a mass difference corresponds to a known PTM not alreadyannotated in UniProtKB/Swiss-Prot, "intelligent" rules areapplied that examine the sequence of the peptide of interestand make predictions as to what amino acid in the peptide islikely to carry the modification.
  4. 4. Protein Sequence to be characterizePeptide Massses
  5. 5. 1.Protein Sequence to be characterizedYou should specify the sequence of theprotein you would like to characterize and forwhich you have determined a set of peptidemasses.
  6. 6. • If the Protein sequence is known• If the Protein Sequence is not known
  7. 7. Enter the UniProtKB/Swiss-Prot ID code (e.g.TKN1_HUMAN)Or the protein accession number (e.g.P20366).
  8. 8. you can enter the sequence of your protein ofinterest, in single letter amino acid code, ineither upper or lower case.The user is required to specify the biologicalsource of the query protein
  9. 9. NoteThe 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.
  10. 10. 2.Peptide MassesEnter the experimentally measured peptidemasses generated from the unknown proteinin the « Enter a list of peptide masses...»textfield, and separate them by spaces, tabs ornew lines.
  11. 11. NOTEYou can copy a list of peptides from Excel orother applications and paste them directlyinto the text field.Avoid using peptide masses known to be fromautodigestion of an enzyme (e.g. trypsin!), orother artefactual peaks (e.g. matrix peaks).Upload a .pkm, .dta or text file
  12. 12. Uploading Peptide MassesUpload 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
  13. 13. Expasy Tools Go to Expasy site: http://www.expasy.ch/tools/#proteomeSelect FinMod Tool
  14. 14. FindMod View
  15. 15. Findmod View
  16. 16. Types of Supported Formats(1) .pkm format(2) Sequest format(3) User Created Files
  17. 17. .pkm Format.pkm format, produced by the Voyager software ofPerseptive Biosystems or the GRAMS software:OP=0Center X Peak Y Left X Right X Time X Mass Difference NameSTD.MiscHeight Left Y Right Y %Height,Width,%Area,%Quan,H/A833.319 2189 833.260833.378 0.016 0 0C0.? 0 762 762854.843 5078 854.769 854.917 0.001 00C0.? 0 3453 3453863.419 5108 863.064 863.775 0.001 0 0C0.? 0 35673567872.402 12519 872.347 872.456 0.002 0 0C0.? 0 11417 11417874.3956730 874.331 874.460 0.002 0 0C0.? 0 3559 3559887.786 5903 887.540888.031 0.003 0 0C0.? 0 4131 4131898.475 3329 898.416 898.534 0.006 00C0.? 0 1377 1377904.366 7432 904.199 904.533 0.001 0 0C0.? 0 55965596955.300 2598 955.229 955.371 0.011 0 0C0.? 0 1089 1089973.845 16689973.749 973.941 0.001 0 0
  18. 18. 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 16689The first line is considered as a comment and is ignored.
  19. 19. User Created FilesAny 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.
  20. 20. NOTEThe upload option only works if you see a browse buttonnext to the text entry field. This should be the case for mostrecent web browser versions, e.g. Netscape 3.0 or higher, MSInternet Explorer 4.0 or higher.
  21. 21. The user can specify whether the program should detect onlypotential PTMs, only single amino acid substitutions or both. The user can choose whether all peptide masses or onlythose that have not been attributed a theoretical peptide inthis process should be examined for potential PTMs resp.single amino acid substitutions.
  22. 22. 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.
  23. 23. MASS TOLERANCEPeptide masses can be specified in ppm (parts per million) orin Dalton.
  24. 24. DIGESTION AGENT (ENZYME)Specify the enzyme or chemical reagent that you used togenerate your peptides (see the corresponding section in thePeptideMass instructions for the available enzymes and theircleavage rules).
  25. 25. MISSED CLEAVAGESIn order to take into account partial cleavages, you can specifya maximum number (0, 1, 2 or 3) of missed cleavage sites tobe allowed. If the maximum number of missed cleavagesentered is 1, all concatenations of two adjoining peptides arealso added to the list of theoretical peptides underconsideration.
  26. 26. SORTING OF PEPTIDES IN THE RESULT TABLESHere you can choose if you would like the peptides to besorted by their mass (from smallest to largest) or by theirchronological order in the protein.
  27. 27. 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.
  28. 28. RESET AND PERFORM BUTTONSOnce 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 tobe reset to their default values, press the Reset button.
  29. 29. OUTPUT FORMATo Headero up to three tables.
  30. 30. 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.
  31. 31. Then the input parameters are listed, followed by an activelink to PeptideMass. This allows the user to perform atheoretical cleavage of the protein of interest.
  32. 32. 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.
  33. 33. FINDMOD OUTPUT } unmodified peptides, modified peptides known in SWISS-PROT and chemically modified peptides
  34. 34. putatively modified peptides predicted by mass differences+ putative AA substitutions {
  35. 35. -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 peptidefragmentation
  36. 36. At the end of the output page the user will find a list of thoseentered matches which did not match in any of the abovetables (if any).

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