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Scaffold

Scaffold. Download free viewer:. http://www.proteomesoftware.com/Proteome_software_prod_Scaffold_download-main.html. download the version matching your operating system follow installation instructions the first time you open scaffold click on “ free Scaffold viewer”

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Scaffold

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  1. Scaffold Download free viewer: http://www.proteomesoftware.com/Proteome_software_prod_Scaffold_download-main.html

  2. download the version matching your operating system • follow installation instructions • the first time you open scaffold click on “ free Scaffold viewer” • no key is required in “viewer mode”, you can open several • copies simultaneously • opening Scaffold takes 1-1.5 minutes, please be patient… • the “viewer” is only for reviewing the results (.sfd files), not for • searching!

  3. start • open the sfd file you received

  4. Samples page * identification probabilities for each protein identified in each biological sample list of proteins identified in the samples annotations if available * instead of the protein identification probability you can display several other information information about the labeled protein

  5. Samples page top toolbar • “View” • you can add or delete different information from the list • lower scoring peptides, which do not reach the threshold you set • low probability hits • annotations • display biological samples or each MS/MS samples • display sequence coverages, number of total or unique peptides assigned to each protein etc. • “Export” • you can export your results into excel spreadsheets • the spreadsheet will contain the report you have chosen and a short description of the • search parameters

  6. Samples page • protein identification probability is color coded; you can set the probability threshold as you like and display • only the hits above this threshold or lower scoring hits as well • consider the trade-off: fewer proteins – fewer false positive hits; • more proteins – more false positive hits • you can sort the data by clicking once at any columns • protein grouping ambiguity indicates if the identified peptides belong to several proteins • (isoforms – see “Similarity” page) • you can search your data for any keywords and filter it for any modifications which were applied at Scaffold search

  7. Proteins page • to reach “Proteins page” choose a protein in the list in the “Samples page” and click on the icon • “Proteins” on the left side • the upper left panel provides you the following information: • protein identification probability, nr. of total peptides assigned to this protein, nr. of unique peptides, • sequence coverage, molecular mass of the protein • choose the protein of interest and the sample in the drop down menu • the upper right panel provides the following information: • sequence of all peptides assigned to the protein chosen in the left panel • mascot ion- and identity scores, if a Mascot search was carried out, modifications, observed and • actual mass, mass differences in Da/ ppm, start and stop amino acids, accession number of similar • proteins in which this peptide is present • the bottom panel provides the following information • sequence coverage of the identified peptides, list of similar proteins, labeling the identified fragment • ions in the spectrum of the peptide chosen in the upper right panel, list of identified fragment ions

  8. Similarity page • to reach “Similarity page” label a protein in the list in the “Samples page” and click on the icon • “Similarity” on the left side • the upper panel provides a vertical list of peptides assigned to this protein, and a horizontal list of • accession numbers of additional proteins, where this peptide is present • the bottom panel provides information about the identification of the labeled peptide similarly as • in the upper right panel in the “protein page” and the assignment of fragment ions as well as a list • of identified fragment ions

  9. Publish page • to reach “Publish page” click on the icon “Publish” on the left side • the right panel contains a short description of the search parameters, suitable for the “ Methods” section (copy/paste), you can generate a peptide or protein report with the icons at the bottom • the left panel contains the same data in form of a table

  10. Statistics page • to reach “Statistics page” click on the icon “Statistics” on the left side • this view provides information about the search process and the evaluation of the spectra

  11. Statistics page • the upper left panel displays : • the number of identified proteins in each sample • the number of identified peptides (≠ number of unique peptides) • the number of identified spectra • the proportion of the identified ones from all generated spectra

  12. Statistics page • the upper right panel displays the relationship between the peptide probability, • the number of identified peptides and the protein identification probability and helps • you setting the filters for protein and peptide identification • note, that one identified peptide, even with 95 % identification probability means • only ~ 50% protein identification probability!!

  13. Statistics paged • the lower left panel displays the relationship between the score values if multiple • search engines were used • each point in the plot corresponds to one spectrum and you can display the • assignment and score values by hovering over each spot with the mouse • dashed lines correspond the filter you set, changing the filter will change the lines • spots colored with red are “good” hits, the ones colored blue are not significant; • changing the filters may change the color of the spots

  14. Statistics page • the lower left panel shows how Scaffold translate the search engines peptide scores • into peptide probability • in case of Mascot a difference of ion and identity score is calculated and then the • number of spectra with each value is defined • two curves are fitted; the lower one shows the distribution of the false positive hits, • the higher one is the distribution of the correct hits • the peptide probability threshold set in the filter is displayed by a dashed line;

  15. Quantify page • to reach “Quantify page” click on the icon “Quantify” on the left side

  16. Quantify page • the bar chart in the upper left panel shows a protein's relative abundance across the • different samples; you can choose any identified protein from the sample in the drop down • menu • the y axis is the normalized spectral count of the protein in each sample where it is present • the normalized spectrum count is calculated this way: the number of identified spectra is calculated in • each sample and averaged, then the number of spectra assigned to one protein is multiplied by the ratio • of the average spectrum count and the number of spectra in that sample • each bar on the x axis is for one biological or MS/MS sample • changing the filter settings may change the quantification

  17. Quantify page • the Venn diagram in the lower left panel shows the number of proteins that have been • identified in each sample category • the diagram can show the overlapping peptides in up to three sample categories

  18. Quantify page • the lower right panel displays the gene ontology terms • the GO terms are arranged to describe biological task, localization • and molecular function of the identified proteins

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