To identify cross-linked peptides from mass spectrometric data,
MeroX/StavroX require a File containing MS-Data (MGF-file
, mzXML-file, mzML-file
or PKL-file) and a FASTA-file.
The MS-file contains precursor information as well as
MS/MS-spectra of the fragmented precursor. The FASTA-file
contains the sequences of all the proteins, that have been used
in the cross-linking experiment. To perform the analysis, StavroX
and MeroX require detailed information about the proteases used, possible modifications, the used cross-linker and technical details of the mass
spectrometric analysis.
From the sequence of the
proteins and the experimental setup, the software calculates all
theoretical possible peptides and compares combinations of those
to the precursor masses of the MS-file. Identified hits are then
scored, comparing theoretical fragmentation of the cross-linked
product to the MS/MS-spectrum.
The sequences of the proteins that were cross-linked (or that are interesting) have to be in a single Fasta file. The mass spectromteric raw data must be converted to a compatible format. This is usually done by the software distributed with the mass spectrometer (or by other software like MSConvert or ReadW). Both files are loaded into the software using the buttons "Load Fasta" and "Load MS-File". Please take time to adjust the settings of the software to the performed analysis (see Settings section).
Fasta-Sequences contain the one-letter amino acid-code, which is also used in the software for the amino acids. Additionally, the software uses a user-defined one-letter code for modified amino acids. For example "M" is used for methionine and "m" is used for oxidized methionine. These definitions can be modified under Settings->Amino acids.
Sequence information always starts with the following character ">" followed by the name of the polypeptide. After a line break the sequence information starts with the first amino acid. After another line break another polypeptide can be entered which starts again with ">" and so on.
The File should be saved as *.fasta but *.txt-files can also be loaded. To distinguish the N-Terminal amine of the protein from the N-Terminal amine of a Peptide after proteolysis, all protein sequences start with a "{"and end with a "}". Peptides start with "[" and end with "]". Opening brackets are defined as one H-atom and closing brackets as OH. The brackets do not need to be defined in the fasta file, but are added automatically by the software.
Fasta files can be downloaded from the uniprot database at uniprot.org. This is the preferred method of retrieving Fasta files as the header information can also be used by MeroX
MGF (Mascot generic Files) can be generated from mass spectrometric raw data. In case of MS/MS-spectra they contain every single MS/MS-spectrum with m/z-value and charge of the precursor.
see MASCOT MGF format for
reference.
MeroX/StavroX reads out:
· m/z-value
· Charge
· Scan number
· retention time
· MS/MS-spectrum
mzXML is on open file format for mass spectrometic data. It was developed at the Seattle Proteome Center.
see mzXML format for reference.
MeroX/StavroX reads out:
· m/z-value
· Charge
· Scan number
· retention time
· MS-spectrum
· MS/MS-spectrum
· MS-level
mzML is a mass spectrometric file format developed by the HUPO Proteomic Standards Initiative and was introduced in 2009.
see mzML
specifications for reference.
MeroX/StavroX reads out:
· m/z-value
· Charge
· Scan number
·
retention time
· MS-spectrum
· MS/MS-spectrum
· MS-level
pkl is a proprietary file format by Waters.
MeroX/StavroX reads out:
· m/z-value
· Charge
· Scan number
· MS/MS-spectrum
To get to the quick setup, go to settings and click on the quick setup button. The quick setup will guide you through the settings for MeroX/StavroX. Firstly, the used protease can easily be selected, so that standard settings for each protease site will be applied. Standard modifications can be selected, some commonly used cross-linkers are available and simplified device settings. After finishing the quick setup wizard, the previous settings will be overwritten. It is advised to save the current settings to a file. The settings window contains a button to restore the default settings.
In the amino acids tab, you can add amino acids as well as modified amino acids. By using the add and remove function you can add or remove an amino acid to the list. You can change the values for name and composition directly in the table.
Name: Set a name for the amino acid you enter.
Code: Select a one-letter-code for the amino acid you want to add.
Composition: Enter the elemental composition for the amino acid. See here how to add or change elements.
Mass: The mass of the amino acid is calculated based on the entered composition.
The specificity of the used protease(s) can be entered into the table. You can use the add and remove buttons to add/remove a protease site. You can change all values directly in the table.
Site: The specificity of the protease is entered as the sequence, with a "?" at the proteolysis-site. "K?" cleaves C-terminal of Lysin e.g. Trypsin. "?D" cleaves N-terminal of aspartic acid e.g. AspN. "ENLYFQ?G" cleaves between Q and G in the sequence ENLYFGQ as does TEV-Protease.
Missed Cleavages: For every different cleavage site a different number of possible misscleavages can be defined. So that only peptides will be calculated, that contain the maximum number of missed cleavage sites.
Blocked by(optional): Defines an amino acid that would inhibit cleavage of the protease if it would be at the position of the "?" in the sequence.
Unspecific Digest: Protease without specificity (e.g. Proteinase K) can also be used with the software. Be aware that using this option may drastically increase complexity of the analysis and therfore the calculation time
Semi-unspecific Digest: One cleavage site (C- or N-terminus) of the calculated peptides needs to be from the specified proteases. The other cleavage can occur unspecifically.
Minimum peptide length: Defines the minimum length of a peptide sequence in a cross-link that the software will include in the search.
Maximum peptide length: Defines the maximum length of a peptide sequence in a cross-link that the software will include in the search.
Unique peptides: If activated, the software will only include unique peptides in the database search. BE AWARE: MeroX does not detect protein families. This option might therefore exclude peptides that are unique to a protein family. If the fasta contains a highly similar isoform of the same protein, most peptides will be excluded from the search.
Isoleucine = Leucine (I=L): For comparision of peptide sequences while testing for uniqueness, isoleucine and leucine are considered equal, as they cannot be differentiated in XL-MS workflows.
Here you can enter static as well as variable modifications of amino acids. You can add or remove a modification by using the add/remove buttons. You can only change the maximum modifications value directly in the table. Static modifications will modify every site, without exception. If you set variable modifications, there will be peptides without the modification as well as peptides containing one or more modifications. All possible combinations will be used, until the number of maximum mofifications is reached.
From: The amino acid that should be modified in the peptides in one-letter code. (e.g. M for methionine)
To: The modified amino acid (e.g. m for oxidized methionine)
Max.mod.: The maximum number of modifications per peptide. If "0" is entered, all amino acids will be modified in all combinations. For example: This would be a theoretic peptide: xxxMxxMMxxx and you enter: from M to m, max.mod.:0 the following peptides would be calculated:
xxxMxxMMxxx
xxxmxxMMxxx
xxxMxxmMxxx
xxxMxxMmxxx
xxxmxxmMxxx
xxxmxxMmxxx
xxxMxxmmxxx
xxxmxxmmxxx
if you enter: From M to m, max.mod.:1 the following
peptides would be included:
xxxMxxMMxxx
xxxmxxMMxxx
xxxMxxmMxxx
xxxMxxMmxxx
Posttranslational modifications: It is possible to search for modifications of a peptide without discrete localization of this modification (as compared to variable modifications). For Composition, enter the modification at the residue (e.g. PO3H for phosphorylation). The modified amino acids are a list of possibly affected amino acids (e.g. STY for phosphorylation). MeroX will consider a maximum of one PTM per peptide.
Additional Information:
A protein N-terminal modification can be entered by modifying the letter '{' to the desired modification (same for C-terminal with '}'). Note that all proteins in the fasta file will be modified then!
This Tab shows a the properties of the currently used cross-linker. You can select another cross-linker from the Drop-down menu. You can add or remove a cross-linker by using the add/remove buttons.
Composition: Enter a composition for the cross-linker. See here how to add or change elements.
Max. Distance: Enter the maximum distance bridged by the cross-linker in Angstrom.
Specificities: The values for specificities 1 and 2 contain all amino acids, that can react with the respective sites. If one site is amine reactive, you might enter "K{" in the spec1-field where 'K' is for lysine and '{' for the N-Terminus of the protein. If the other side is sulfhydryl reactive, you should enter "C" for cysteine in the spec2-field.
Ignore consecutive Peptides: With most cross-linkers, it is not possible to distinguish between a cross-link of two consecutive peptides (in the sequence of the protein) and a dead-end (type 0)- cross-link. Therefore this option can be used to excluded consecutive peptides from the search.
MeroX/StavroX can identify dead-end cross links. To do so the check-box for dead-end cross-links (type 0) must be activated. Dead-end cross-links usually occur by reaction with water. You can change the molecule the dead end reacted with in the "dead-ends reacted with"-field. It is also possible to add several Molecules separated by semicolons
Intrapeptidal cross-links (type 1) can also be identified, activating the respective check-box.
For MS-cleavable cross-linker: Specific MS/MS-fragments of the cross linker can be entered in the table on the right, by using the add button.
Essential fragments are required for proper identification of a cross-linked spectrum during RISE,RISEUP and Proteome-wide modes). Non-essential fragments are not required for the proper identification. These ions will still be identified and labeled in the spectra.
Report-ions Cleavable cross-linkers with two cleavage sites (such as DSBU, DSSO or PIR) can produce small ions when two fragmentation events occur. The ion mass is independent of the cross-linked peptides and can only be found in spectra of cross-linked peptides. These reporter-ions are not used for the identification algorithms, but a have minor effect on scoring.
Additional Information:
For the DSBU cross-linker a specific loss is known that occurs only on intrapeptidal cross-links. This constant neutral loss (CNL) can also be modified in the settings.
A zero-length cross-linker without a mass shift is entered by leaving the composition field blank. MeroX is going to calculate the mass difference as 0.
A disulfide cross-link of two cysteines to cystine can be included as '-H2'.
This window contains all information required for a proper analysis using StavroX or MeroX.
Precursor precision: (Default: 5 ppm) Defines the deviation that a precursor can have to the calculated mass of crosslinked peptides, to be recognized as cross-link-candidate.
Precursor mass recalibration: (Default: 0 ppm) Precursor masses can be recalibrated by a fixed value (in ppm) to correct for mass drifts of the mass spectrometer.
Fragment ion precision: (Default: 10 ppm) Defines the deviation that fragment ions can have to the calculated fragment ion mass, to still be identified. Precision can be entered in Da or ppm for high resolution spectra.
Fragment ion mass recalibration: (Default: 0 ppm) Fragment ion masses can be recalibrated by a fixed value (in ppm) to correct for mass drifts of the mass spectrometer.
Lower/Upper Mass Limit: (Default: 1000/6000 Da) Defines the lowest/highest precursor masses.
S/N-Ratio: (Default: 2x) In Fragment ion comparison, only peaks with a signal/noise ratio above the defined value will be compared to theoretical fragment ion masses. The noise is calculated iteratively for 5 up to 10 different sections of the spectrum until only signals are left that have a higher signal to noise ratio than defined.
Ion types: (Default: b,y) Select all ion types that should be analysed (a,b,c,x,y,z,z' or z''). The z' and z''-ion types can other types. In the Settings file you will find a section that begins with "IONTYPE". This section defines the z' and z'' as a standard. These can be modified only in the settings file, not from the graphical user interface!
Minimum nr. of fragments per peptide (Default: 3) Set the minimum required number of different fragment ions per peptide in a cross-link.
Ignore detected charge states (Default: Deactivated) MeroX will ignore the charge state of the precursor and test all defined charge states (only available if activated) for every precursor.
Minimum Charge (MS1) (Default: 2) Select the minimum charge state of a precursor that is required for further processing.
Deisotoping: (Default: activated) For high resolution spectra it is possible to deisotope the spectra prior to analysis. This will improve the analysis as 1) isotopic signals are removed from the spectrum and 2) only possible fragment with the charge of the signal will be compared resulting in less false-identification of signals.
Negative Mode (Default: Deactivated) In case, MS-analysis was done in negative mode, check this box.
Precursor mass correction (Default: Deactivated) MeroX will correct wrongly assigned precursor masses due to wrong monoisotopic peak picking algorithms during conversion. For mzML and mzXML, MS1- information is used for correction. For mgf-files, all possible mass shifts are tested for each spectrum. Recommended to be activated for mzML and mzXML.
Save spectra to temp folder (Default: deactivated) MS-spectra will be saved to a local folder to reduce RAM usage for larger files. For mzXML and mzML files, temporary files are always written to a temporary folder.
Losses: (Default: option 2) you can specify, whether neutral losses of fragment ions should 1) not be included, 2) only be included, if an ion without a loss has been identified and 3) ion losses of all possible ions should generally be included in the search.
Specific Loss: (Default: empty) You can specify a neutral loss, that might occur often in your experiment, by entering its composition and checking the box.
All peptide combinations that match the mass of the precursor are considered as cross-link candidates and are scored as such. This mode does not require MS-cleavability and is therefore applicable mainly to non-cleavable linkers such as DSS/BS3 or BS2G. This mode is computationally more extensive and might not give conclusive results or crash when many proteins are in the protein database or many amino acids are considered for cross-linking.
Reporter Ion Scan Event Mode is based on scanning the spectrum for possible cross-linker reporter ions. From these fragments possible peptides are identified, matching the cross-link mass. The algorithm should only be used with high resolution spectra that are deisotoped, preferably by MeroX or by a third party software.
Max. Missing Ions (RISE&RISEUP-Mode): It is possible to identify a spectrum with an incomplete fragmentation pattern. You can set the maximum number of ions that can be absent from the pattern. But at least one fragment for each peptide must be identified!
In this mode, MeroX analyses the spectrum for pairs of signals corresponding to cross-linker fragments. From these pairs, MeroX infers possible peptide masses and compares the peptides to the spectrum. In case of a match (e.g. peptidescore > 10), the peptide database is searched for a matching second peptide. This peptide is also scored against the spectrum. If both peptides have a peptidescore above the set threshold, the candidate is considered as a cross-link and scored again.
The RISEUP mode is a combination of RISE- and Proteome-wide mode (with minimum peptide score of 2). All combinations that would pass the RISE-filter or the proteome-wide filter are considered as cross-linked and are scored.
Prescore: (Default: 10%) For prescore calculation, the precursor region is filtered out of the spectrum and only high intensity signals (>10% of the remaining base peak) are used. The prescore equals the coverage of fragment ion intensity in this filtered spectrum in %.
FDR cut off: Based on a target-decoy analysis, a false
discovery rate is estimated for each candidate. Score cut offs
for different false discovery rates can be estimated in the decoy
analysis window. The FDR cut off is applied while finalizing the
result file. Any candidate above the false discovery cut off will
then be removed from the final results. FDRs are calculated
independently for interprotein, intraprotein, dead end and
intrapeptide cross-links using
FDR = (TD-DD)/TT
where TT is the number of target-only matches, TD the number mixed target-decoy matches and DD is the number of decoy-only matches above a certain score threshold.
Score cut off: (Recommended: 0 - 50) All cross-link-candidates with a score higher than the defined threshold will be saved to the result file. All other candidates will not be considered. To include all candidates this value has to be -1. This setting is useful for larger analyses, where the score cut off can be applied to reduce memory requirements, as fewer candidates need to be saved.
Score settings: (Default: max) It is possible to adjust the speed of the scoring algorithm and to not score at all. The different Score settings differ in the number of shifted spectra the unshifted spectrum is compared to. (2, 4, 6, 8). More spectra (up to 8) will increase the reliability while reducing the speed of scoring.
Exclude internal linear ions: (Default: Deactivated) For MS-cleavable cross-linkers cleaving at the cross-linking site and leaving the free peptide (e.g. CDI), secondary fragmentation events are indistinguishable from linear fragments and should therefore be excluded from scoring.
invert sequences: All peptide sequences of the given fasta
database will be inverted to obtain false positive
identifications.
shuffle sequences: All peptide
sequences of the given fasta database will be shuffled to obtain
false positive identifications.
shuffle sequences
but keep protease sites: All peptide sequences of the given
fasta database will be shuffled while keeping the amino acids of
protease sites in place, to obtain false positive
identifications.
Include cRAP database: For analyses with single proteins and activated RISE-Mode or RISEUP-Mode, the decoy analysis will not be precise, as only a few wrong peptides will be identified from the decoy database. In these cases it is advised to increase the size of the sequence database. In MeroX the cRAP database (theGPM.org) is included to increase the size of the decoy database. The cRAP database contains the sequences of commonly occuring contaminants in MS-analyses.
Here you can set the number of digits, that should be shown in tables as well as spectra, for deviations, precursors and fragments.
Default protein name type: If Uniprot headers are used, MeroX can show the Gene name (e.g. GAPDH), the Uniprot ID (e.g. P04406), the Entry-name ID (e.g. G3P_HUMAN), the full protein name or the complete fasta header.
All file dialogs will start at the last opened folder. You can choose whether this folder will be stored for each file type separately or not.
Site numbering Cross-link sites can either be displayed relative to the peptide-index (e.g. K4-K1) or relative to the protein sequence index (e.g. K432-K25).
The color scheme of all spectra in MeroX/StavroX can be customized. It is possible to select between two different coloring modes. Either the signals are colored by peptide or by ion type. The different colors can be selected by clicking on the box indicating the used color for a signal type. A Color chooser dialog will pop up and a new color can be defined. The original settings can also be retrieved. The coloring scheme is stored in MeroX.ini in the directory of the executable and will be loaded everytime MeroX starts, so it is independent of different experiments.
The used cross-linker as well as the amino acids are entered as formula and the masses are calculated by the program based on the given element masses. To enter compositions follow these simple rules:
· Elements with one character (CNOSP...) are written in CAPITAL
letters.
· For elements with two characters (Na, Li) the
first one must be a CAPITAL letter and the second must be lower
case!
· Elements are followed by their number (none
means 1)
· all elements after "-" will be subtracted
examples: C6H12O6, CH3CH2OH, Na2CO3, -H2
Element definition:
The masses for the elements are defined in the file "properties.mxf" or in the user-defined *.mxf files. (or *.ssf for StavroX) The List does not contain the masses of all elements or isotopes of those. So it might be necessary to add a mass. Therefore you need to change the *.mxf while StavroX is not running.
To add an atom these rules must be followed:
· Line must be in between "ELEMENTS" and "END"
· Enter
the line with no spaces!
· Element name (max. 2
characters!) is followed by ";" and the mass of the atom with up
to 8 digits
· Elements with one character (CNOSP...) are
written in CAPITAL letters.
· For elements with two
characters (Na, Li) the first one must be a CAPITAL letter and
the second must be lower case!
The section containing the element information in the settings file looks something like this:
ELEMENTS
H;1.00782503
D;2.01410178
T;3.01604927
O;15.9949146
C;12.00000000
N;14.003074
S;31.9720707
P;30.9737615
Li;6.0151223
F;18.9984032
Na;22.9897697
Si;27.9769265
Cl;34.9688527
K;38.9637069
Br;78.9183376
I;126.904468
END
After the analysis is finished, a window is on top that shows a bar chart and a line graph. The bar chart plots the number of candidates identified in a certain score range to the score range. The line graph shows the dependency of FDR at different score cut offs
Both Graphs can be visualized for all kinds of cross-link types: as well as the peptide score distribution.
Both figures should help to estimate the quality of the score for your experiment. Blue bars represent the number of candidates from your dataset. Red bars represent the number of false-positive candidates from a decoy dataset. This decoy dataset is calculated, e.g. by inverting the sequences you supplied in the fasta file and performing exactly the same analysis as with the right sequences. This inversion of the sequence can only lead to false-positives.
In a "score-region", where candidates from the real data set are significantly enriched, cross-links are more probable. Like in the figure above for scores over ~60.
In the top of the window, the score regions for different FDRs are given. Cross-links with a score of ~60 and above have an FDR below 1%. The fields on the bottom allow you to calculate the FDR for a certain score region and vice versa.
The Main Window consists of four panels.
· The candidate
table (top left)
· The unique cross-link site table (top
right)
· The cross-link position and fragmentation panel
(bottom left)
· The spectrum panel (bottom right)
You can change the sizes of the panels by dragging and dropping the separators.
You can quickly save your analysis and changes (check boxes and miscelleneous field) by pressing [Ctrl+S] or save it to a different file by pressing [Ctrl+Shift+S].
Since version x.6.5 it is possible to calculate the euclidean distances of the unique cross-linking sites that were identified using structural information from one or more pdb files. It is possible to either load severeal downloaded pdb files or enter several pdb-Codes in the textbox. In the latter case, the software will download the pdb-file automatically, which will take a bit longer. Time consumption will dependent on the speed of internet connection.
The candidate panel holds all the candidates that have been
identified from your dataset. It is possible to use the shortcuts
[Ctrl+A] and [Ctrl+C] and paste the data to excel
or similar software. If you select one candidate from the list by
clicking on it or using the keys up/down, details of this
cross-link are shown in the two lower panels
To sort
the data by any of the columns, simply left click on the
column header.
To hide or show columns, right
click on the table header and select the columns to show. It is
also possible to filter for text and search for a
mass.
Text filter: enter the filter text in the
text field above the table.
Mass search: enter
mass and deviation in the fields above and push the search
button.
you can read out the following information:
| Nr. | consecutive numbers of rows in the table |
| score | The best score calculated for any combination of the cross-link sites in the two peptides. |
| Q-value | statistical measure of confidence |
| Pepscore | Scores of individual peptides when compared to the spectrum as a cross-link to an unknown second peptide. |
| m/z | Mass to charge ratio |
| charge(z) | Charge of the precursor |
| measured Mass | Mass of the singly charged precursor M+H+ |
| calculated Mass | theoretical singly charged mass of the cross-link |
| deviation in ppm | The deviation of theoretical and measured mass in parts |
| peptide sequence | Sequence of the peptide, to both ends brackets have been added that represent hydrogen ('[') and hydroxyl (']') |
| Protein | defines the protein, the peptide is derived from |
| from/to position in protein | defines the start/stop position of the peptide in the protein |
| Sites | indicates the cross-linked positions in the cross link candidate that gave the best score. |
| rank | If more than one candidate was identified for the same spectrum, this number gives the rank of the selected candidate with 1 being the best. |
| scan | The Scan that is given in the Mgf file |
| RT | Retention time of the precursor in a possible LC run |
| misc. | A Field that can be used to write any text. The content will be saved upon closing the Window. |
| select | You can select the cross-link candidate to simplify analyses. Selection status is saved when the window is closed. You can filter for selected candidates with "TRUE" |
Filtering of rows: MeroX/StavroX support a basic regular
expressions filtering. Most regular expressions will work in the
software.
- | for OR combination
- space or
& for AND combination
- ^ for beginning of line
- $ for end of line
- \+5 for selecting all candidates
with a charge of +5
Advanced Filtering: It is possible to filter the result table in an advanced mode. It is possible to only filter for values within a distinct row. It is also possible to filter for all results above or below a certain threshold.
This panel contains the combined results of the result table. All cross-linking candidates, pointing to the same site are condensed into one unique cross-link site. This means, that all charge states, peptides of different length or differentially modified peptides are combined. To see, which candidates have been combined into a unique site, select the Highlight checkbox. This will highlight all combined candidates in the result table.
The P%-value for the best scoring candidate will be listed in this table. If the site annotation is ambigous, all possible sites will be listed, separated by "/". The P%-value indicates the likelyhood of the shown site combination.
Filtering and changes within the candidate table will immediately affect the candidate table.
This panel holds a table, that contains all possible combinations of cross-link sites of the two proteins with the corresponding score and probability (P%). The probability is calculated only from site determining fragment ions and their corresponding intensities. This only includes branched fragment ions, that do contain parts of the cross- linker or the cross-linker plus the second peptide Selecting a row will refresh the 'Fragmentation View' and the spectrum panel and show the details of this specific combination of cross-linked sites. To get more details on this cross-link, use the 'Details of Cross-link'-button. This will open the details window. If you click on the fragmentation view, a popup will show with a more detailed version of the fragmentation view. The fragmentation view is explained in the Details Window section below.
If you think a cross-linking site combination was wrongly assigned you can change the identified cross-linked site in the candidate table by double-clicking on the preferred site combination in the detail panel.
To show the decoy analysis window, use the 'Show Decoy Analysis'-button.
This panel shows the MS/MS spectrum for the identified cross link. Identified peaks are labeled (y-ions in blue, b-ions in red, precursor ions in green and if different types were identified in one signal, it is shown in magenta - colors can be changed in the GUI settings) You can zoom by dragging and dropping in the window or by using the mouse wheel. Unzooming works with a double click. If you double click on one of the axes, only this axis is going to be unzoomed. You can also use the right click menu to unzoom or reset zoom.
The cross-links identified with StavroX and MeroX can be visualized easily using xVis (4). If the Fasta file used for the analysis contains standard Uniprot fasta headers, it is sufficient to save the xVis file and upload it to xVis. If custom fasta headers are used (even if it concerns only a single protein), you also have to export a protein length file to be used instead of automatic uniprot determination of protein length (This will be done automatically for Results generated with StavroX3.6 or MeroX1.6 or higher). In xVis you can filter for false discovery rates (type 'FDR' and select below threshold). Please also read the instructions on the xVis site on how to use xVis.
This window contains all information calculated by MeroX/StavroX to validate the cross-link at a certain site. Several cross-link candidates can be opened simultaneously and will be shown as tabs. You can also drag and drop those tabs to a new window to compare spectra side-by-side. Panel 1, 2, 3 and 4 can be printed out on one sheet, summarizing the evaluation of the candidate. The graphic can also be exported as EPS, EMF, JPG or PNG.
Panel 1 - Spectrum Panel This panel shows the MS² spectrum for the identified cross-link. Identified peaks are labeled (y-ions in blue, b-ions in red, precursor ions in green and if different types were identified in one signal, it is shown in magenta) You can zoom by dragging and dropping in the window or by using the mouse wheel. Reseting the zoom works with a double click. If you double click on one of the axes, only the zoom of this axis is going to be reset. You can also use the right click menu to unzoom or reset zoom. Hovering over signals will highlight them in the fragment view and the table of identified signals.
Panel 2 - Deviation Diagram The deviation of the identified signals is plotted as a function of m/z below the spectrum. This diagram can be hidden via the right click menu.
Panel 3 - Fragmentation View The sequences of the two peptides are shown and a line between the two represents the cross-linker. Marks on both sequences represent ions resulting from fragmentation of the precursor. The color of the mark correlates to the intensity of the ion in the spectrum according to the legend below the figure. The charge of this fragment ion is given on the right hand side. Hovering over the marks will highlight the signals in the spectrum and the table of the identified signals. This view can be hidden via the right click menu.
Panel 4 - Cross-link candidate information This panel summarizes information about the candidate, like mass, charge, involved peptides and cross-linker as well as score information. It will be printed along with panel 1 and 2.
Panel 5 - Table of identified ions This table shows all
identified ions with its intensity, relative intensity,
calculated and measured mass, the deviation, the ion type,
charge, which peptide they are derived from and a possible
neutral loss.
To sort the data by any of the
columns, simply left click on the column header.
It is also possible to filter for text and search
for a mass.
Text filter: enter the filter text
in the text field above the table.
Mass search:
enter mass and deviation in the fields above and push the search
button.
Selecting entries from this table will highlight
the corresponding signals in the spectrum as well as in the
fragmentation view.
Panel 6 - Theoretical fragment table This table shows the sequences of both peptides, C-terminal and N-terminal fragment ion masses of all possible charges. It also shows the masses of neutral losses of the fragments shown.
Panel 7 - View settings panel Here you can set the settings for this Window. You can select between the 6 ion types known to MeroX/StavroX (a,b,c,x,y,z). It is also possible to only show identified signals of one peptide, or without neutral losses. You can also exclude unidentified signals or precursor signals.
MeroX can plot the interaction network detected in the analysis as a node-network. Use the filter to select the cross-links you want to visualize in the network. Tip: Select the cross-links you want to show and then filter for 'true' (without quotes). In the main window click on "View->show Network Graph"
Several small tools have been added to MeroX:
