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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
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!

start

open the sfd file you received

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

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

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

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

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

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

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

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

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!!

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

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;

Quantify page

to reach Quantify page click on the icon Quantify on the left side

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

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

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