SpectroDive 7 - Leviathan
User Manual
Table of Contents
1
System Requirements ........................................................................................................... 3
2
General Information .............................................................................................................. 3
2.1
3
4
Supported Instruments ................................................................................................. 3
Getting Started ...................................................................................................................... 4
3.1
Getting SpectroDive ...................................................................................................... 4
3.2
SpectroDive Activation .................................................................................................. 4
3.3
iRT Kit ............................................................................................................................ 4
3.4
Assay Panel Guidelines .................................................................................................. 5
3.4.1
Biognosys Assay Panels ......................................................................................... 5
3.4.2
External Assay Panels ............................................................................................ 5
SpectroDive ........................................................................................................................... 5
4.1
Prepare Perspective ...................................................................................................... 6
4.1.1
Panel Management ............................................................................................... 6
4.1.2
LC Calibration ...................................................................................................... 10
4.1.3
Exporting Method Files ....................................................................................... 11
4.1.4
iRT Refinement .................................................................................................... 11
4.2
Review Perspective ..................................................................................................... 12
4.2.1
Tree Filtering ....................................................................................................... 14
4.2.2
Tree Grouping...................................................................................................... 14
4.2.3
Saving/Loading Experiments ............................................................................... 14
4.2.4
Experiment Editor ............................................................................................... 14
4.2.5
Panel Matching.................................................................................................... 15
4.3
Visualization ................................................................................................................ 15
4.3.1
Run Node Plots .................................................................................................... 15
4.3.2
Peptide and Fragment Plots ................................................................................ 17
4.4
Report Perspective ...................................................................................................... 20
4.4.1
Normal Report ..................................................................................................... 21
4.4.2
Pivot Report......................................................................................................... 21
4.5
Quality Control Perspective ........................................................................................ 21
4.6
SpectroDive Command Line Mode.............................................................................. 22
5
Support ................................................................................................................................ 22
6
References ........................................................................................................................... 23
Page 2 of 23
1
System Requirements
Minimal
Operating System:
CPU:
Hard drive:
Memory:
Software:
Windows 7, x86
Intel ® Core CPU, 2.7 GHz (quad-core) or similar
50 GB free space
4 GB
.NET 4.5
Recommended
Operating System:
CPU:
Hard drive:
Memory:
Software:
Windows 7 or higher, x64
Intel Core i7 4770, 3.4 GHz (octa-core) or similar
50 GB free space, solid state drive (SSD)
16 GB or more
.NET 4.5 or higher
2
General Information
SpectroDive™ is a Biognosys’ proprietary software for automated MS method set-up, signal
processing, and analysis of MRM and PRM experiments. It features:
 Automated MS method set-up, signal processing and analysis
 Integrated iRT calibration
 Powerful peak picking
 Immediate and intuitive data visualization
 Customized reporting
 Fully automated quality control
2.1
Supported Instruments
Thermo Scientific Q Exactive
Thermo Scientific TSQ Vantage
AB SCIEX QTrap 4000
AB SCIEX QTrap 5500
Page 3 of 23
3
Getting Started
3.1
Getting SpectroDive
The SpectroDive software can be downloaded at https://shop.biognosys.ch/spectrodive. After
successful registration you will receive a download link together with a license key that is
needed to activate your software.
Besides the software, additional reference material including raw data and the corresponding
assays containing accurate iRT can be downloaded.
3.2
SpectroDive Activation
When you start SpectroDive for the first time, you will be asked to activate your software. You
should have received a license key in a separate email (please also check your spam folder). If
you do not have a license key yet, you can obtain one by registering yourself and your academic
institution at https://shop.biognosys.ch/spectrodive.
If your computer has access to the internet, activation will be automatic once you have pasted
your license key into the SpectroDive activation dialogue. In case your SpectroDive computer
does not have an internet connection or the connection is blocked by a Firewall, you can also
activate your software using email. The respective instructions will appear after a few seconds
if online activation was not successful. Save the registration information file on your computer
and send this file to [email protected]. You will receive a license file usually within 1
business day. To activate SpectroDive using a license file, click on the “Browse License File…”
button in the SpectroDive Activation dialogue.
3.3
iRT Kit
To enable full potential of SpectroDive, we developed the iRT Kit that is recommended to be
spiked into each sample before the measurement. The iRT Kit contains a mix of non-naturally
occurring synthetic peptides. Based on these peptides, SpectroDive determines iRT and
calibrates important parameters for peak detection. Using SpectroDive with the iRT Kit
translates into better results:




Setup scheduled MRM/PRM Easy setup of scheduled methods on your LC system
Smart in-run parameter calibration guarantees quick and robust analysis
Higher quantitative accuracy by supporting optimized ion current extraction in PRM
Automated quality control by monitoring LC and MS performance (Figure 1)
For more information about the iRT Calibration Kit, please visit http://www.biognosys.ch/iRT/.
Page 4 of 23
Figure 1 Quality control perspective of SpectroDive based on the peptides in the iRT
Kit. Chromatography, mass spectrometer performance and analysis can be
monitored over time using several performance indicators.
3.4
Assay Panel Guidelines
Assay panels can be imported when starting an experiment analysis by selecting a Biognosys’
kit file or importing an external assay panel.
3.4.1 Biognosys Assay Panels
Biognosys offers high-quality commercial kits for MRM analysis. You can read more about it by
going to http://shop.biognosys.ch/spectrodive/ and then selecting MRM Assay Panels. It is also
possible to generate custom panels for your needs by contacting us via [email protected]
3.4.2 External Assay Panels
If an external assay panel is to be used, you can either use your existing pipeline to generate
assays or use the Prepare perspective of our DIA software, Spectronaut. Please refer to the
Spectronaut manual for detailed information on generating spectral library using shotgun
analysis. The advantage of using Spectronaut for creating your MRM or PRM assays is that in
few easy clicks you will have generated a high-quality assay panel for SpectroDive. If needed,
you can further refine the iRTs of the peptides within SpectroDive as described in the iRT
Refinement section (4.1.4)
4
SpectroDive
Use the test data provided on our webpage or acquire your own MRM or PRM runs with the iRT
Kit and a corresponding assay panel to start testing SpectroDive.
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Figure 2 Powerful cross runs visualization of SpectroDive, here for 24 MRM LC-MS runs.
4.1
Prepare Perspective
The prepare perspective in SpectroDive allows you to export instrument-specific, scheduled or
unscheduled, method file for your assay panel. To facilitate this, it provides features that let you
manage assay panels, specify the calibration to be used for a scheduled experiment, and finally,
export the method file based on the panel and calibration information.
4.1.1 Panel Management
For MRM/PRM experiments, an assay panel representing a transition list is necessary to
analyzing your data. SpectroDive provides several features to allow flexible management of your
panels as underlined below.
4.1.1.1 Importing Panels
You can import panels into SpectroDive by selecting “Import New Panel…” link and then
selecting either a Biognosys’ kit format file or a compatible spreadsheet in a plain text format.
The panel imported from a spreadsheet must follow a few formatting rules (see reference
material for an example):


Plain text, in a separated value format (tsv, csv)
The header needs to specify your columns
If a text-based file is used, a Kit Import Manager (Figure 3) will be displayed which will try to
auto detect the column names. If there are new column names SpectroDive can remember
those as soon as you assigned them once. SpectroDive will ask you for every new column
association whether or not you want to store it as recognized synonym for this column. In case
you accidentally saved a wrong association, you can remove the user defined column synonyms
in the settings (settings perspective -> “Column Recognition”).
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Figure 3 Kit Import Manager allows you to import your text based panel into
SpectroDive by matching the column headers to the SpectroDive specific column
requirements.
SpectroDive will warn you if you forget to assign a mandatory or recommended column. Once
the panel is successfully imported, it will be displayed in the Panels list. You can organize your
panels by creating new folders by right-clicking. The minimal requirements on the panel are two
columns, the precursor ion m/z and the fragment ion m/z. Although an analysis would already
work with only the two minimally required ones, it is highly recommended to provide the
information listed below in order to achieve the best possible results.
4.1.1.2 Panel columns
A SpectroDive panel represents a MRM/PRM transition list. Please use the following information
to achieve the best possible results.
Q1 (Required)
The in silico calculated m/z of the peptide precursor ion. Please do not round this number.
Q3 (Required)
The in silico calculated m/z of the peptide fragment ion. Please do not round this number.
iRT (Highly Recommended)
The peptide retention in the reverse phase chromatography as expressed with the normalized
retention time iRT2. If accurate iRTs are provided this will speed up the analysis and increase the
quality of your results (sensitivity, specificity, accuracy). In order to derive iRTs for your peptides
use the Biognosys’ iRT Kit spiked into your shotgun proteomics runs.
RelativeFragmentIntensity (Highly Recommended)
The relative peptide fragment ion intensity expressed as a percentage of the most intense
fragment ion. Please report the raw number without the percentage sign. This information
improves the limit of detection.
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StrippedSequence (Recommended)
The stripped amino acid sequence of the peptide excluding any modifications. Please only use
the single letter code for the 20 standard proteinogenic amino acids. This information is used
for labelling and scoring of your fragment ions in SpectroDive. Further, it is included in the
automatically generated unique ID for your precursor if necessary.
PrecursorCharge (Recommended)
The peptide precursor ion charge as an integer. This information is used for labelling your
precursors in SpectroDive and automatically generating a unique ID for your precursor if
necessary.
FragmentType (Recommended)
The peptide fragment ion type. Usually this is “y” or “b”. This information is used for labelling
and scoring of your fragment ions in SpectroDive .
FragmentNumber (Recommended)
The peptide fragment ion an integer. This number should be between 1 and the length of your
peptide in amino acids minus one. This information is used for labelling and scoring of your
fragment ions in SpectroDive .
FragmentCharge (Recommended)
The peptide fragment ion charge formatted as an integer. This information is used for labelling
and scoring of your fragment ions in SpectroDive .
FragmentLossType (Recommended)
Allows you to specify the fragment loss-type (e.g. NH3 or H2O). This is mainly used for labelling
your fragment ions in plots.
ModifiedSequence (Recommended)
In case your peptide is modified use this column to specify the amino acid sequence including
modifications. The modified sequence should be constant for one unique precursor. This
information is used for labelling your precursors in SpectroDive and automatically generating a
unique ID if necessary. The modified sequence is not used for fragment calculation but only for
grouping and displaying purposes. The actual content can therefore be in any desired format
(e.g. _[ac]M[ox]AGILC[CAM]K_).
ProteinId
The ID of the protein the peptide is derived from. This information is used for labelling your
peptides. SpectroDive provides filtering capabilities in the review perspective including filtering
for the protein ID and also grouping by proteins.
Workflow
SpectroDive supports SPIKE_IN, LABEL, INVERTED_SPIKE_IN, and LABEL_FREE workflows as
described below. If a workflow is specified in the panel using these keywords, SpectroDive will
always use that workflow when analyzing runs matched to that panel regardless of the default
workflow specified in the settings. The default workflow in SpectroDive is SPIKE_IN.
LABEL_FREE: No reference peptides used. One channel per peptide.
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SPIKE_IN: Stable isotope standards (SIS) are spiked into your sample and you expect the
heavy channel to be always present. SpectroDive uses the SIS signal to detect the target
signal, but False Discovery Rate (FDR) analysis is done based on the target signal.
LABEL: Peak detection and FDR analysis is done on both heavy and light channels.
INVERTED_SPIKE_IN: Same as SPIKE_IN but light channel is used as the reference.
Decoy
It is possible to specify explicit decoys in your panel. The value of the column should simply be
True or False, where True indicates decoy.
UserGroup
A column for additional user information that can be used for grouping and filtering.
For
an
exemplary
panel
please
visit
the
SpectroDive
https://shop.biognosys.ch/spectrodive and download the test data set.
webpage
on
4.1.1.3 Splitting Panels
You can split an existing panel into smaller panels by right-clicking on a panel and selecting Split.
This will open a Split Panel form where you can specify the number of Transitions per split. If
you have any explicit decoys present in the panel, they will be carried over into each split.
SpectroDive will distribute the assays evenly over the iRT range across the splits.
Figure 4 Split panel allows you to systematically
split a large panel into smaller panels that can
then be measured independently by creating a
method file for each
4.1.1.4 Generating Sub-panels
A sub-panel can be generated from an existing panel by right clicking on the panel node. This
will open a form (Figure 5) in which you can select specific precursors and/or transition groups
for creating a new sub-panel. If using a Biognosys MRM Assay Panel (like PlasmaDive), the
selection is only allowed at the precursor level.
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Figure 5 Generating sub-panel gives you a finer control over creating
a new panel which is a subset of the source panel.
4.1.2 LC Calibration
SpectroDive allows selection from the N most recent runs, 10 by default (Settings  SpectroDive
 Panels  Calibration History), that were analyzed in the Review perspective for iRT-based
retention time calibration. You can also load calibration directly from a raw file by clicking on
the Calibration from Raw File link. The calibration file will be used for calibrating the iRT values
of your assays to generate a scheduled acquisition method.
You can edit a specific calibration file to select which of the iRT peptides are to be used for
calibration (Figure 6).
Figure 6 Edit calibration allows you to reject specific iRT peptides and/or review information specific to the
calibration run
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4.1.3 Exporting Method Files
You can export a method file for your panel by selecting the right instrument. If making a
scheduled experiment, SpectroDive will automatically schedule your assays based on the
specified calibration file and window size parameters. The concurrent transitions plot (Figure 7)
gives you an overview of the scheduling of your panel. Adjust the retention time window for
scheduling in the field ‘Window (min)’ in order to not exceed a maximum number of concurrent
transitions to avoid an unfavorable signal-to-noise ratio of the data (e.g. assuming a constant
cycle time of 2.5 seconds, one should not exceed maximal 250 concurrent transitions since this
may lead to a dwell time of lower than 10 ms).
Figure 7 Concurrent transitions plot gives you an overview of the scheduling of your panel.
4.1.4 iRT Refinement
SpectroDive also allows you to refine iRT values of a given panel based on specific runs. This can
be useful at an early stage of panel development or if migrating the panel to a very different
chromatographic setup. To do so, you must have your panel imported into the panel repository
in the Prepare perspective. When you are ready to export a method file for the panel, select “iRT
Refinement” in the field ‘Run Type’ instead of “Analysis.” This will create a special method file
for acquiring runs meant for refining your panel. SpectroDive will automatically use a wider
window by default when exporting a method meant to be used for iRT refinement. To
accommodate this, it will use only two transitions per peptide.
Once, you have acquired the runs using this method file, load them in the review perspective
without manually assigning a panel. SpectroDive will automatically detect the correct panel. At
this point you can right-click on the experiment tab and select “Refine iRT” which will open the
iRT Refinement dialog (Figure 8). Please note that the iRT refinement will only be done for
peptides which are manually accepted in the review perspective.
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Figure 8 iRT refinement allows you to refine the iRT values in your panel.
4.2
Review Perspective
By default, SpectroDive starts up in the review perspective. This perspective allows a manual
reviewing of your MRM/PRM data while the complete experiment is processed in the
background.
To load an experiment, click on the “Load Raw Data…” link. The experiment setup dialogue will
appear (Figure 9). First, load one or several runs. You can then assign the panel manually or let
SpectroDive match panels automatically from the panel repository in the Prepare perspective
(Importing Panel).
While setting up your experiment, you can select an analysis schema for your experiment. You
can generate new customized schemas in the settings perspective. The analysis schema allows
you to specify a certain set of parameters that you want to use for your analysis. In most cases
the default schema will produce the best results.
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Figure 9 Experiment setup dialogue of SpectroDive. A raw file is required.
After you click “Start” SpectroDive will switch to the review perspective and start the analysis.
First, in every run, SpectroDive will try to detect the iRT Kit and calibrate analysis parameters for
each run based on your specific data. The evaluation of signals from the iRT Kit allows fast data
processing, easy setup of scheduled methods, improves specificity and enables automated
quality control. As such, it is highly recommended to spike in iRT Kit in all the samples. Browsing
your data will be possible a few seconds after the initial calibration process is finished (Figure
10). At the bottom left, you can see a progress bar informing you about the overall status of the
analysis. After the analysis has finished the number of unique precursors, peptides and proteins
identified will be shown in the bottom right for a Qvalue cutoff of 0.01 (corresponds to a false
discovery rate of 1%).
SpectroDive determines identification on a wide range of scores.
Figure 10 Review perspective of SpectroDive. The lower right panel shows the correlation of relative fragment ion
intensities between measurement and the values provided in the panel.
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4.2.1 Tree Filtering
Using the filtering system implemented in SpectroDive, one can apply one or several filters on
the review data tree. Select a filter from the dropdown menu and set the filter criteria. The filter
is now marked as selected within the dropdown menu. To combine filters, select a different filter
and define the value that should be applied. A precursor has to apply to all selected filter in
order to by shown in the review tree.
A special filter criterion is the “User Group” filter. This value can be set during the library import
by selecting a specific column as “User Group”.
4.2.2 Tree Grouping
If you right-click on the experiment tab, you can apply different grouping strategies on the
experiment tree. By default, precursors are grouped by the respective precursor window. Other
grouping possibilities are



Protein: Allows a grouping via protein ID. The Parent node shows overview
information about this group.
Panel (default): Shows all precursor according to their associated assay panel.
User defined Group: Similar to the tree filtering the user group, as defined during
spectral library import, can be used for a more custom grouping.
4.2.3 Saving/Loading Experiments
Saving and loading of a complete analysis is supported. Saving an experiment will also store
manually integrated peaks, comments, excluded fragment ions and whether a peak was
manually accepted or rejected. To save an experiment right-click on the experiment tab and
select “Save As…” in the context menu. The most recently loaded or saved experiments are
shown in the “New Experiment” page of the SpectroDive review perspective. A saved
experiment in SpectroDive will contain the full analysis information and can therefore get very
large in size but does not require the raw file or panel to be available anymore.
4.2.4 Experiment Editor
Experiment editor (Figure 11) allows you to add new annotation to the experiment which will
be visible in the Report perspective as user defined columns. For instance, you can add a column
that annotates the disease state of each run. Also, you may change the run order here.
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Figure 11 Experiment editor allows you to add custom run based annotation and
change the order of your runs.
4.2.5 Panel Matching
SpectroDive will automatically detect the correct panel from the panel repository and match to
it. If for some reason the panel is not auto-detected, it could be because the matching
percentage was below a certain threshold. By default, SpectroDive expects 80% of the
transitions present in the panel to be matched to a run for it to count as a successful match. You
can lower this threshold in Settings  SpectroDive  Panels  Matching Fraction (Figure 12).
However, you can also manually force SpectroDive to match to a panel by assigning it in the
Experiment Setup.
Figure 12 Settings perspective of SpectroDive allows you to control analysis related and general settings.
4.3
Visualization
The review perspective of SpectroDive comes with a wide variety of powerful visualization
options for different levels of your experiment review tree.
4.3.1 Run Node Plots
On the run node level, one can access different plots which give you detailed information
about the calibration, run meta information and cross run performance.
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4.3.1.1 iRT Calibration Chart
This chart shows the calibration of iRT peptides spiked in to your sample. If no iRT Kit was
used, SpectroDive will not be able to calibrate your run and this plot will not be displayed.
Figure 13 iRT Calibration Chart showing the linearity of RT ~ iRT for the iRT peptides.
4.3.1.2 Run Overview & Overlay Chart
The Run overview chart (Figure 14, top panel) shows the extracted chromatograms of all
targeted peptides of this run. The Run overlay (Figure 14, bottom panel) combines the
information of all runs of your experiment in one plot for better insight into instrument stability.
You can right-click on these plots and select “Show Point Labels” to annotate the peaks with the
matching peptide sequence. Also, it is possible to toggle the x-axis between retention time and
iRT scale.
Figure 14 The RT Run Overlay Chart shows the total ion current of all runs in your experiment in a single plot, giving
insight into instrument stability.
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4.3.1.3 Run Overview
Run overview gives you important information pertaining to the quality of the current run
ranging from panel identification success to instrument acquisition related information.
Figure 15 Run overview gives you an overview of important run specific parameters.
4.3.2 Peptide and Fragment Plots
There are several different visualization options for the different peptide and fragment node
levels in your review tree. These range from XIC visualization to more score-centric and crossrun profile visualizations. The later ones are only available in multi run experiments and disabled
for experiments containing only one run or peptides that are only targeted in one run.
4.3.2.1 Iontrace Chart
The default plot on Elution Group (EG), Transition Group (TG) and Transition (T) level (Figure 16).
This plot shows the extracted ion chromatogram of the selected peptide. The plot contains the
iontraces for all fragments present in the panel. Additionally, the expected retention time is
marked (black dotted line) and the currently selected peak for this peptide (green area). On
Elution Group and Transition Group level the coloring of the fragments is based on its expected
relative intensity ranging from red (expected most intense fragment) to blue (expected least
intense fragment).
On Transition level, this plot only shows the selected fragment in color and all others in gray.
Figure 16 Ion trace for the peptide EGNTFLDLSVR++. The color coding of the fragments indicate an overall good
correlation to the expected fragment intensities.
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4.3.2.2 Iontrace Sum Chart
The Iontrace Sum chart (Figure 17) shows the selected peptides quantitative information. The
iontrace shown is the sum of all transition iontraces that qualified for quantitation.
Figure 17 Sum of ion traces for all transitions of the peptide EGNTFLDLSVR++.
4.3.2.3 Intensity Corr Chart
This plot gives you detailed feedback about the accuracy of your fragment intensity prediction.
The predicted values correspond to the relative intensities provided by the spectral library
indicated in red. The black lines correspond to the relative measured intensity of each fragment
of the predicted peak.
Figure 18 The fragment intensity correlation plot for a given peptide precursor. The plot indicates a very good
correlation between the expected relative intensities (red) and the measured intensities of the library fragments.
4.3.2.4 Alignment Chart
The Alignment chart (Figure 2 and Figure 19) allows you to visualize the extracted ion
chromatograms of a single peptide across all of your runs. You can directly change integration
boundaries in the plot to quickly manually validate a quantitative peptide profile.
Page 18 of 23
Figure 19 The alignment of the peptide SFLTYIK++ across 3 runs. The x-axis is automatically changed to iRT to reduce
chromatographic variance. The axis can be changed to retention time by right clicking on the plot and un-selecting
the “Use iRT Scale” option.
4.3.2.5 iRT EG Overlay Chart
This plot allows you to show all sum ion traces of your selected peptide from all runs in a single
chromatogram. The axis is automatically converted to iRT but can be changed to reflect the
actual retention time.
Figure 20 The iRT EG Overlay chart for the peptide IILDLISESPIK++. The 3 XICs correspond to the sum XIC of one peptide
in the 3 different runs loaded for this experiment.
4.3.2.6 Fragment Intensity Alignment
Similar to the Alignment Chart, this plot gives detailed information about the peak picking
stability across several runs (Figure 21). The different bars show the relative abundance of each
fragment across multiple runs. Using this plot, one can quickly identify a wrongly picked peak by
the sudden change in the color pattern. The plot can also be changed to reflect actual intensities
instead of relative abundance by right clicking on the plot and un-selecting “Normalize”.
Figure 21 The fragment Intensity alignment for a peptide containing 4 fragments. Each fragments relative intensity
compared to the total peak height is indicated using a differently colored bar. The peptide was targeted in 3 runs.
Page 19 of 23
4.3.2.7 Cross Run RT Accuracy
Similar to the Fragment Intensity Alignment chart, the Cross Run RT Accuracy plot allows you to
quickly validate the peak picking across several LC-MS runs. The x-axis shows colored bars that
correspond to the peptide in different runs. On the y-axis one can see the retention time in iRT.
The height of each box corresponds to the peak width the start and the end iRT according to the
y-axis. The line through the middle of the box shows the apex retention time in iRT while the
blue colored boxes in the back show the total XIC extraction width. The colors of the bars again
corresponds the relative intensities of the measured fragment ions. The bar with the green
background is the currently selected node. The black, dotted, horizontal line corresponds to the
expected retention time in iRT. You can hop to any other node by clicking on the colored bar.
Figure 22 The Cross Run RT Accuracy plot for a peptide measured in 3 different runs. The multi-colored bars
correspond to the detected peak with the colors encoding the relative fragment intensities and the upper and lower
boundary of the bar corresponding to the start and end of the peak integration boundaries.
4.4
Report Perspective
After your experiment was successfully loaded in the Review perspective you can switch to the
Report perspective to export detailed information (Figure 23). The Report perspective provides
you with great flexibility allowing you to specify and save your own report schemas. In the
schemas you can specify which data should be exported and define column names such that
they fit your needs.
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Figure 23 Report perspective of SpectroDive. Experiment, LC-MS run, Elution Group (precursor), Transition Group
(labeled precursor), Transition (Fragment) centric reports can be generated.
4.4.1 Normal Report
A normal report represents a typical spread sheet report where each row corresponds to the
type of report being displayed. There are five different types of reports: Experiment, Run, Elution
Group, Transition Group and Transition report. Lower level reports can include information from
higher level reports but not vice versa. For instance, in a fragment report, each row corresponds
to a distinct fragment.
4.4.2
Pivot Report
Pivot report allows you to compare the selected row and its cell values across all the runs in your
experiment. Each selected cell value’s column header will specify the parent run, one for each
run. The row labels, representing the Meta information, will be only present once in the report.
If a selected cell value does not pass a selected filter criteria then it will be reported as “Filtered”.
4.5
Quality Control Perspective
The quality control perspective of SpectroDive (Figure 1 and Figure 24) is based on the peptides
in the iRT Kit. Chromatography, mass spectrometer performance and analysis can be monitored
over time using several performance indicators. Every successful analysis is stored to the quality
control perspective. SpectroDive automatically detects various instruments and will create a
separate quality control history for each of them. In case you have more than one instrument of
the same type it might be useful to rename them manually. Additional folder structures can be
made according to the established quality control testing performed in a specific laboratory.
Please note that if no iRT peptides are detected in a given run, there will be no QC data recorded
for that run.
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Figure 24 A QC plot showing the observed retention time of iRT peptides over 9 different runs.
4.6
SpectroDive Command Line Mode
Additionally to the visual pipeline mode, SpectroDive is also capable of running the pipeline from
command line. To run SpectroDive in command line mode you simple call the SpectroDive.exe
file using the following parameter.
-r
-d
-s
-o
-n
-rs
Adds a run (*.raw or *.wiff) to the experiment.
Adds all wiff/raw files of a specified directory to the experiment.
[OPTIONAL] Selects a settings schema (the schema must already exist)
If this schema is not present, the default settings are used
[OPTIONAL] Specifies a output directory for the reports
If this parameter is not present, reports will be generated in
%Appdata%/SpectroDive/Results
[OPTIONAL] Specifies a name for this experiment
[OPTIONAL] Selects a report schema (the schema must already exist). If this
schema is not present then the default schema will be used.
An example:
SpectroDive.exe –r C:/Users/bgs/A_D150526_SQC-Yeast_MsMRM_R03_T0.raw
5
Support
Please post your message on the Biognosys helpdesk at http://www.biognosys.ch/helpdesk/ or
send us an email at [email protected]
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6
1
2
References
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