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

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. open the sfd file you received start

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

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