The following article contains a list of frequently asked question relating to views and displays in Scaffold Q+S. For specific questions not covered in our documentation we are available by telephone Monday through Friday from 8 AM to 5 PM PST. Our toll free number is 1-800-944-6027. Additionally support can be contacted via email at email@example.com.
Views and Panes
What does the Median Density Chart tell me?
The Median/Density Chart provides a visual method to judge how much the proteins are differentially expressed at the category level.
Category Density Function (Density Probability): The thin line is the approximated, empirical density distribution of all the experimental data included in each category for a specific protein. Scaffold Q+S estimates the distribution using a Kernel Density function based on a finite number of measurements. In our case, the measurements considered come from all the spectra in all samples belonging to a particular protein in a specific category. The function also allows combining data from different samples into one overall distribution.
Sampling distribution of the Averages (Median Probability): To help ascertain a protein's differential expression, Scaffold Q+S includes a plot of the sampling distribution of the averages estimated by a statistical procedure called bootstrapping. The fewer data points available, the less defined the average will be and the distribution will appear more sprawled. The area underneath the sampling distribution of the averages is shaded according to the coloring assigned to each category.
What do the Samples View Display Options mean?
Protein Identification Probability: This displays Scaffold’s calculated probability that the protein identification is correct. Large difference in this value are significant whereas differences of just a few percent are probably not.
Number of Assigned Spectra: The total number of spectra that matched to all peptides in the protein.
Log2 Fold Change: This will display the log2 of the fold change between abundance of each multiplexed tag and the base multiplex tag. For instance, if a protein is upregulated by a factor of 2 between two multiplex samples the log2 fold change value will be 1.0 This value will always be zero for the base multiplex tag.
Fold Change Ratio: Ratio between the measured value for a quantitative intensity to the reference quantitative intensity.
Normalized Intensity Value: The normalized Log2 of the intensities.
Coefficient of Variation: Normalized measure of the dispersion of the distribution of the quantitative values.
How does the Sample Wide Fold Changes pane work?
Displays the log2 fold change of all proteins in the sample. Proteins are ordered from most negative fold change to most positive fold change. The selected protein is highlighted in yellow. If the Log2 fold change of the selected protein is close to zero, then a yellow protein indicator may not be displayed on the graph.
What do the Intensity Weighting and Average CVs graphs mean?
Intensity Weighting: The Intensity Weighting graph plots both the weight and median log2 summed raw intensities of all proteins against their standard deviations. For example, if a protein has 7 peptides with a median log2 summed raw intensity of 26 and a median weight of .82, the corresponding points will be plotted in purple and green, respectively. Once Scaffold Q+S plots all proteins in the sample, it plots a line along each of the measurements to display a line graph.
Average CVs: The Average CVs graph is designed to suggest whether the data in each sample might be suspect due to uneven coefficient of variation. If one sample has significantly deviant coefficient of variation from the other samples, then it has a higher likelihood of being bad or suspect data. However, if all coefficient of variations seem relatively constant, then you can estimate fairly little error in the data.
How do I interpret the Raw Intensity Distribution graphs?
The Raw Intensity Distribution Pane provides three graphs for informing how to organize and normalize the data:
Pre/Post Normalization: The Pre/Post Normalization graph shows the amount by which Scaffold Q+ normalizes each sample. At the bottom of the Pre-Normalization graph, each sample displays the percentage of missing spectral data. Quantitative analysis of samples with high percentages of missing data might be unreliable.
Raw Quantifiable Intensity: The Raw Quantifiable Intensity graph displays, for all proteins in the selected Primary Sample, the distribution of intensities. Ideally, this should resemble a normal distribution. The Bin Resolution slider on the left designates the number of bins, or sections of the data with equal widths, to display. For instance, a Bin Resolution of 10 will provide ten bars in the histogram and weight the bars according to the number of data points in the bar. A Bin Resolution of 35 will divide those ten bars into thirty-five bars and then plot the histogram accordingly. Toggle whether to view the log2 intensities or the raw intensities by clicking the Log checkbox.
Signal to Noise: The Signal to Noise graph displays a histogram based on each individual peak intensity in a sample as compared to the largest peak intensity in the selected primary sample. The height of the bars in the graph correspond to the number of measurements at each percentage. Again, the width of the bars is dependent on the Bin Resolution as described above.
What do the Normalized Intensity Scatterplots show me?
Each graph compares two Quantitative Samples. The Primary and Secondary samples are selected using drop down boxes above the graphs.
Protein Scatterplot: Displays the relative reporter ion log2 normalized intensities, or the light, heavy or medium labels for SILAC, for all proteins in the sample. The X-axis is primary sample quantitative value and the Y-axis is secondary sample quantitative value. An optimal 45° reporter ion correlation line is drawn over the scatter plot and represents a 1:1 ratio between the selected quantitative samples.The selected protein is highlighted in yellow. If the selected protein is up regulated then the yellow dot will be located subset beyond the optimal correlation line in the upper left corner of the scatter plot. If the protein is down regulated it will be below the optimal correlation line.
Multiplex Scatterplot: Displays the relative reporter ion log2 intensities, or the light, heavy or medium labels for SILAC, for all spectra in the sample. The X-axis is primary sample quantitative value and the Y-axis is secondary sample quantitative value. The peptides assigned to the selected protein are highlighted in yellow. If the selected protein is up regulated then the trend for the yellows subset of the scatter plot should have a slope greater than one. If the protein is down regulated the expected slope would be less than one. Normalized values can be used by checking the box at the top of the graph. An optimal 45° reporter ion correlation line is drawn over the scatter plot and represents a 1:1 ratio between the selected quantitative samples
What are the differences between the three Protein and Peptide Level Graphs?
The Protein Level Charts and Peptide Level Charts display relative quantitation information for the selected protein or peptide for each Quant Sample Category in the experiment. Click and drag to zoom in on the graph. A single left click will zoom out one step. Error bars will be displayed when enough data is missing or suspect.
Three different types of plots can be selected
Box Plot & Histogram: Displays the relative medians and ranges for the fold change values of the protein and peptides selected.
Trend Line Plot: Displays the relative fold change values for each peptide in the selected protein or each spectra in the selected peptide. You can select an individual peptide or spectra by hovering over a data point. The relative change line will be highlighted in bold and the peptide will be selected automatically in the peptide pane.
What does it mean if I have "No quantifiable peptides" in a protein?
This means that in the protein selected, there are no spectra to quantify on or no quantitative information about the protein in the sample
>What are non-unique peptides?
Non-unique peptides are peptides that match more than one protein identified in the sample. By default, these peptides are not used for quantification because Scaffold Q+ does not automatically know how to distribute their quantities among the proteins that share the peptides.
Why do I see some peptides in Scaffold that aren't listed for the same protein in Q+S?
If a peptide does not have any quantitative data associated with it, it will not show up in Q+S. This happens most often with SILAC data, as some peptides may not get labeled with the Light/Medium/Heavy labels. The parent program (Distiller, Discoverer, or MaxQuant, for example) may also filter peptides differently than Scaffold does upon loading the data.
It is important to note that Scaffold Q+S also only chooses one spectra per peptide for quantification unless the spectra have different charge states. For instance, peptide AISEILEK might show 2 spectra in the Proteins View of Scaffold Q+S because one of the spectra has a +2 charge and the other spectra has a +3 charge.