MASH Suite

User Manual

© 2013 Ge Research Group. All rights reserved.

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Table of Contents

1) Introduction

2) Installation

3) Toolbar

4) Workflow and Parameters

5) Spectrum View

6) Experiment Window

7) Sequence Table / Theoretical Data

8) Organizing Your View

9) Error Handling

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Introduction

MASH Suite provides a versatile and user-friendly interface for high-resolution mass

spectrometry (MS) data analysis. It is based on the THRASH algorithm and Decon2LS open

source code to process mass spectral data. MASH Suite is a multifaceted software package

which is designed to provide a host of functions for effective MS data processing, interpretation,

visualization and presentation.

MASH Suite contains several visualization modules, written in C# in the .NET

programming environment, as well as backend libraries (DeconEngine) written in C++. MASH

Suite can provide the user with several tools to facilitate data processing, interpretation,

visualization and presentation options. MASH Suite is able to handle high-resolution data files

in a user-friendly environment that allows the user to perform the pre-processing, processing,

and post-processing of their mass spectrometry data in a single interface. By including all the

tools needed to complete the workflow for top-down, middle-down and bottom-up proteomic

experiments, users are able to analyze high-resolution MS data in an effective, user-friendly

environment. MASH Suite is a free software package which is available for download upon

request.

Installation

MASH Suite can be installed on a Windows operating system, and Microsoft .NET

Framework 4 must be installed before the program can be installed. In addition to this, Xcalibur

2.0.7.1044 or more recent versions must be installed in order to open Thermo Finnigan Raw data

files. Contact the Ge Research Group (ge2@wisc.edu) to get access for installation.

After running the installation file, a shortcut (Figure 1) will be created on the user‟s

desktop.

Figure 1. MASH Suite icon

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Toolbar

The toolbar (Figure 2.) is located at the top of the program and consists of six buttons: File, Edit,

View, Tools, Experiments and Help. The functions of these buttons are enumerated below.

Figure 2. MASH Suite toolbar

File:

New Experiment (Ctrl + N) – opens another experiment file (.RAW). The first file opened

generates a Spectrum View window, an Experiments window, and a window with the file

pathway as the title. Additional files can immediately be added, and a list of the current

files can be seen in the Experiments window. Multiple copies of the same file can be

opened, and the work specifically done on each of them can be saved separately.

Open Sequence – opens a sequence file (.sec) in the Sequence Table.

Open Saved Results – opens and resumes saved project files.

Save Results – saves current project as an XML file. This file can then be opened either by

right clicking the open, selecting „Open with,‟ and choosing MASH Suite, or choosing the

Open Saved Results option under File in the Toolbar.

Print Results – prints the results.

Exit – exits the program.

Edit:

Copy Results – copies the list of peaks after data processing. The results can be paste into

any document.

Copy Chart – copies the chart. This chart can then be pasted into any Word or PowerPoint

document.

Copy Table – copies the sequence table information as an image that can be pasted into any

document.

View: shows or hides the following windows: Toolbar, MassList, Status Bar, Mass List and

Sequence Table.

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Experiments: These options are used when handling multiple experiment files. After

multiple experiment files are uploaded, they can be arranged to be simultaneously viewed using

the following tools:

Tile Vertical – rearranges the Experiment Windows so that they are both visible with one

above the other.

Tile Horizontal – rearranges the Experiment Windows so that they are both visible side by

side.

Close All – closes all currently open Experiment Windows.

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Workflow and Parameters

The Workflow and Parameters window (Figure 3) contains two tabs: workflow and

parameters, and parameters (shown below). The workflow section contains five subsections

(Add Experiments, Process Your Data, Analyze Your Data, Quantitate Your Data, and Report

Your Analysis). The parameters section allows the user to make any necessary pre-processing

modifications to before extraction.

Figure 3. The Workflow and Parameters window and Parameters Window

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Workflow and Parameters – Add Experiments

Figure 4. Load Experimental Data

Load Experimental Raw Data (Figure 4) - There are THREE ways to do this

1. Use New Experiment under File in the Toolbar,

2. Choose from Load Experimental Data under the Workflow and Parameters Command

Explorer.

3. Use the shortcut control + N.

When opening a file from the Workflow and Parameters Command Explorer, the user needs to

specify the type of file to open. There are two possible types of files that can be loaded and each

is described in the following sections:

Thermo Finnigan Raw Data File – generated by a Thermo mass spectrometer with a .RAW file

extension. These files cannot be opened using MASH Suite unless the user has Xcalibur

2.0.7.1044 installed.

mzXML Data Files – not generated by a mass spectrometer, but through programs that have

access to the native files. mzXML is one of the most commonly used file formats for storing

hyphenated-MS data.

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Show Spectrum View

After a certain data file is opened, the Spectrum will be displayed as shown below (Figure 5).

If the spectrum window is closed, it can be displayed by choosing a specific experiment and

clicking Show Spectrum View under Add Experiment.

Figure 5. The Spectrum View window showing the spectra and the Experiments

window showing the experiment file of the displayed spectra

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Define Parameters

MASH Suite allows users to define a variety of parameters for high-resolution MS data

processing and analysis (Figure 6).

Figure 6. The pop-up window for users to define parameters.

S/N Threshold (Signal to Noise Threshold) – sets the acceptable threshold ratio of signal to noise

that will be used as the criteria to determine whether an experimental peak cluster can be added

to the list of identified peaks. Noise is determined by the minimum intensity of the most

abundant isotopomer, and signal of each peak cluster is determined by the intensity of its most

abundant isotopomer. To reduce the false positive rate, a signal to noise intensity threshold is

established to separate biological data from interference generated by the instrument. The

threshold is calculated by the following two steps.

Step 1: threshold1 =

Step 2: threshold =

; if

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The S/N threshold therefore defines the lowest S/N to be considered in peak-picking process.

Typically S/N = 3.0 or 1.5 is used based on the confidence level needed. Setting a lower S/N

threshold enables lower intensity peaks to be detected that would otherwise be undetected, but

also increases the chances of producing false positives. Using the New Match (Figure 7)

function in Spectrum View, the user is able to selectively modify the S/N threshold to scan

certain sections of the M/Z spectrum for lower intensity peaks without producing false positives

at other sections of the spectrum (Details about the New Match function will be elaborated on

Page 29.)

Figure 7. The New Match function in the Spectrum View window

Maximum Charge – sets the maximum charge state that a peak can have in order to be added to

the list of potential proteins. It limits the highest charge state of the peaks to be considered in

peak-picking process to help reduce the false-positive discovery rate. For bottom-up MS, the

charge state of fragments ions are usually significantly lower than the fragments generated from

top-down MS partially because of the small sizes of the fragments. A maximum charge state of

50 is usually sufficient for bottom-up and top-down MS; for very large proteins a higher

maximum charge state value should be used.

Minimum Fit (%) – sets the minimum fit score between the theoretical peak cluster and observed

experimental peak cluster needed for the experimental peak to be added to the list of identified

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peaks. This parameter is also known as minimum reliability, and the value is determined using a

least-squares fitting procedure that generates a percentage accuracy between the experimental

and theoretical peak clusters. The calculation is as following:

Fit score =

; in which is the i

th isotopic point‟s intensity of the observed isotopic

pattern and is the ith

isotopic point‟s intensity of the theoretical mass.

Minimum M/Z – sets the minimum M/Z threshold below which no spectral data will be

considered possible proteins.

Maximum M/Z – sets the maximum M/Z an experimental peak cluster may have to be added to

the list of possible peaks.

Workflow and Parameters – Process Your Data

Figure 8. Peak Retrieval function for data pre-processing

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Pre-processing – Peak Retrieval – this function populates the Peak List with all the possible

peaks without characterizing them with theoretical profiles.

(Note: The current MASH Suite version only provides the option of THRASH to process the

data. We are working on the other option.)

Figure 9. The green bar showing progress of data processing

Figure 9 shows the Spectrum View window of a mass spectrometry dataset being analyzed. The

partially filled green bar at the bottom of the screen shows the progress of extraction, which acts

as an indicator for how close the data is to being completely processed.

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Mass List – After the mass spectrometry data are analyzed, resulting theoretical profiles are generated and

scored according to how well they match with the observed data. All the data fields for this

information have been generated in a table that can be sorted.

Figure 10. An example of a Mass List

# – number assigned for the peak cluster.

Monoisotopic – sum of the masses of the atoms in the molecules, using the most abundant

isotope for each atom. This is calculated after THRASH deconvolution.

Abundance – the absolute intensity of the most abundant isotopomer in a peak cluster.

M/Z – the mass to charge ratio of the most abundant isotopomer in a peak cluster.

Charge – charge state of the peak cluster.

Score – fit score. Measured as a percentage and calculated based on how well the

theoretical profile matches the experimental profile.

Most Abundant – mass value of the most intense isotopomer within a isotopic peak cluster

Ion – type of ion. In full MS mode, “MI” is assigned to the ions that have the same

monoisotopic mass as the reference amino acid sequence. Other ions will be displayed as

“MI +/- the mass difference”. In MS/MS mode, according to the reference sequence, the

ions can be b, y, c, or z, followed by the position of the first amino acid from the N-terminal

sequence.

Theoretical (Da) – mass value of the theoretical monoisotopic value, which is the mass

value of the monoisotopic isotopomer in the theoretical peak cluster.

Error (Da) – difference in daltons between the Theoretical (Da) and the Monoisotopic

value.

Error (ppm) – difference in parts per million between the Theoretical (Da) and the

Monoisotopic value.

Formula – the chemical formula generated for the peak cluster based on the averagine and

the theoretical profile.

Quantitation – the summation of experimental intensities for an isotopic cluster. This can

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be done through the Calculate Quantitation Scores in the Quantitate Your Data section of

the Workflow and Parameters window.

Peak List –contains a list of every isotopomer peak (Figure 11), regardless of whether a

match was identified for the peak.

Figure 11. An example of a Peak List from a dataset

# – number of the individual peak, assigning the peak starting from the smallest M/Z value.

M/Z – the mass to charge ratio of an individual isotopomer. This is the x-axis in the

spectrum graph.

Intensity – the absolute abundance of an individual isotopomer. This is the y-axis in the

spectrum graph.

FWHM – the full width at half maximum of an individual isotopomer.

S/N – the signal to noise ratio of an individual isotopomer.

Mass pairs – contains a list of discovered mass pairs. These values are generated after the

user has input mass difference and Err (Da) values in the toolbar of the Experiment Window and

pressed Search (Figure 12). This tool can be used to search for potential peptide modifications

and fragments. When a mass pair is selected, the corresponding peaks are highlighted as purple

diamonds.

Figure 12. An example of a Mass Pairs list

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# – number assigned for the mass pairs, starting with one for the smallest monoisotopic

mass value.

Mass1 – monoisotopic mass value of the first experimental peak cluster.

Mass2 – monoisotopic mass value of the second experimental peak cluster that differs from

Mass 1 by the mass difference set by the users.

Charge1 – charge state of the first mass.

Charge2 – charge state of the second mass.

Delta Mass – mass difference of the mass pairs. This value is restricted to a value that is

within the error range of the mass difference that was determined before the Search.

Modification – if available, displays the modification assigned based on information from

the Sequence Table.

Theoretical – theoretical monoisotopic mass of a matched ion fragment from the Sequence

Table.

Err (ppm) / Err (Da) – the difference in mass (reported in either ppm or Da) between the

theoretical peptide fragments and experimental data. Modifying the error threshold can be

done in the Tolerance Information section of the Sequence Table window.

Users can sort the lists based on their preference by click the item to be sorted in the lists.

(Example: Mass List sorted by Monoisotopic Mass)

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Toolbar (Figure 13):

Figure 13. Toolbar of the Experiment window

Select/Unselect – checks/unchecks all the identified spectral peak clusters in the Mass List.

Delete – deletes the selected peak cluster from the peak cluster list. This can be useful if the

program has identified peak clusters that are not real peaks.

Copy – copies all the peak clusters, where they can be pasted in other document files.

Show – shows the currently selected spectral clusters in the Spectrum View by putting a purple

diamond symbol above the most abundant peak of the peak cluster for each selected peak cluster.

Print – prints out all the information in the Mass List for the currently selected experiment.

Add Note – adds a note to the selected spectral cluster in the Mass List.

Search – generates a list of mass pairs according to the Mass and Err(Da) parameters:

- Mass – value used for the mass difference between spectral peak clusters to generate the

list of mass pairs.

- Err(Da) – value used for an acceptance region around the Mass value to identify mass

pairs that are not exactly the Mass value.

This feature can be used to identify peptide fragments if the mass difference between the

fragments is known. The mass search results are shown in the Mass pairs list discussed above.

Processing - Mass Deconvolution – this function allows the user to select the method for

detecting the peaks. Users can choose either THRASH or MS-Deconv, which have slight

differences in their algorithms. (We are working on the MS-Deconv option.)

Database Search Wizard – The peak cluster identifications generated using the THRASH

algorithm can be sent to a proteomics database to be matched with molecular profiles. If the

experiment was run in full MS mode, entire proteins can be identified, and if the experiment was

run in MS/MS mode, peptides can be identified that belong to the precursor ion that was used.

(We are working on this option)

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Workflow and Parameters – Analyze Your Data

Figure 14. Import Protein. Peptide Sequence under Analyze Your Data

Import Protein.Peptide Sequence

Importing peptide or protein sequence data for analyzing your data can either be done

through importing a file (the file extension is .seq), manually inputting the sequence, or pasting

the sequence data from a source (e.g. uniprot.org).

To manually input or paste the sequence information, select Import Protein.Peptide

Sequence and then select Paste Your Sequence (Figure 14). The text box shown below will then

appear. Delete “CHANGEME,” and enter the amino acid sequence using standard amino acid

abbreviations. Select OK after inputting the sequence information to update sequence.

(Note: Manually pasting the information from a database website automatically removes all

numbers and irrelevant information from the selection that is copied.)

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Experiment Mode

MASH Suite can be run in top-down, middle-down or bottom-up experimental modes (Figure

15). Under the top-down mode, the user can specify whether the experiment is an MS or MS/MS

Experiment. If an MS/MS Experiment is chosen, the user can specify the fragmentation method

to be CAD or ECD Fragmentation. If the Middle-down mode is selected, the user is prompted to

specify the enzyme that was used (trypsin is set as the default).

Figure 15. Selection of Experimental Mode

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Workflow and Parameters – Quantitate Your Data

Figure 16. Quantitation of the peak abundance

Calculate Quantitation Scores – It takes several minutes to generate the quantitation score. The

score is assigned according to average intensity of the average intensity of the top five peaks in

the isotopic peak cluster (Figure 16). Users are able to change the number of peaks taken for

calculating the quantitation scores through the Parameters window. There is a loading bar located

at the lower right corner of the screen that shows the progress of quantitation scores calculation.

The generated scores can be seen in the Mass List under Quantitation.

Show Quantitation Chart – creates a bar graph that replaces the Spectrum graph in the

Spectrum View window (Figure 17). The x-axis of this graph is a list of the identified ion

fragments in the order of increasing M/Z value, and the y-axis is the absolute abundance of these

fragments.

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Figure 17. A quantitation bar graph showing the relative abundance of four proteoforms from 3 top-

down datasets with error bar showing the standard deviation. The bar graph can be directly exported

to PowerPoint or any other word processing software and edited.

Show Quantitation Table – creates a table that replaces the Spectrum graph in the Spectrum

View window. This table shows the statistical relationships between different selected

conditions, groupings and subgroupings.

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Workflow and Parameters – Report Your Data

Figure 18. Selection of several ways to report MS data

Report Files – allows users to save their results into a file, print the results file, copy the results

to a clipboard, or create a peak depot file (Figure 18).

- Save Results File – saves the current workspace of the user into an XML file. Opening

saved files can be done through File Open Saved Results, or using the shortcut

Ctrl+Shift+O.

- Print Results File – prints the workspace of the user.

- Copy result to clipboard – copy the information of the peak clusters in the Mass List to

clipboard, the result can be paste to any document.

- Create peak depot file – export the Mass List into an Excel file.

Report Images – allows the user to copy their sequence table or copy their spectrum image

(Figure 18).

0

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Parameters – Experiment Data

Figure 19. Experiment data in the Parameter window

XCalibur File – displays the file pathway of the most recently opened raw data file (.raw).

Clicking the button next to the file pathway opens the user‟s directory to the location of that file,

and allows the user to open another experiment file.

Experiment Type – default is Full MS, but can be changed to CAD Fragmentation, ECD

Fragmentation, or Enzymatic Digestion.

Sequence File – displays the file pathway of the uploaded sequence file (.seq). Clicking the

button next to the file pathway opens the user‟s directory to the location of that file, and allows

the user to upload another experiment file.

Sequence Data – default is CHANGEME, and can overwritten with sequence information by

either inputting the sequence manually, or pasting the sequence from any source.

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Parameters – Ion Assignments

Figure 20. Ion Assignment in the Parameters window

Charge – default is M, and can be changed to (M+H). This is the charge type.

Tolerance – default is 1. This is the tolerance mass value for ion assignments.

Tolerance Type – default is Da, and can be changed to (ppm).

Mass Type – default is Monoisotopic, and can be changed to Average. This changes the value

used for the experimental profiles that would then be matched with ion fragments.

Draw b/y Ions – default is true.

Draw c/z Ions – default is false.

Draw b*(NH3) Ions – loss of NH3 from b ions. Default is false.

Draw bo(H2O) Ions – loss of H2O from b ions. Default is false.

Draw bp(HPO3) Ions – loss of HPO3 from b ions. Default is false.

Draw yp(HPO3) Ions – loss of HPO3 from y ions. efault is false.

Draw bpa(H3PO4) Ions – loss of H3PO4 from b ions. Default is false.

Draw ypa(H3PO4) Ions – loss of H3PO4 from y ions. Default is false.

Print All – default is false. Enabling this function allows users to use the Print Results File

under Report Files in the Report Your Data section of the Workflow and Parameters window.

EnzymeType – default is None. The enzymes available for selection are: Trypsin,

Chymotrypsin-high specificity, Chymotrypsin-low specificity, Lys C, Lys N, CNBr, Glu C

(bicarbonate), Glu C (phosphate), Asp N, Arg C, Pepsin (pH 1.3), Pepsin (pH > 2), Proteinase K,

and Thermolysin.

Missed Cleavages – default is 5. This is the maximum number of missed cleavages allowed in

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order for an ion assignment.

Parameters – Mass Parameters

Figure 21. Mass Parameters in the Parameters Window

Maximum Charge – default is 50. This is the maximum charge that an experimental peak

clusters can be selected and matched to a theoretical profiles.

Minimum Fit – default is 60. This value is a percentage, and it is minimum fit score for a

theoretical profile to fit an experimental peak cluster for the profile to be included in the

Identification List. Setting higher values would reduce the number of actual matches, and is a

trade-off between possibly losing valid matches and generating data that are more accurate.

Averagine Threshold – default is 0.5, and is the value that sets a limit to how much the

theoretical chemical formulas generated can deviate from the the averagine values. This

parameter is used to limit the number of isotopic points to be shown. Isotopic points of low

intensity region will not be shown.

Mass Range – default is 250-2000, and is the M/Z region that is scanned for isotopic peaks.

Increasing the Mass Range increases the amount of the time it takes to complete extraction.

Averagine Table – default is C4.9384 H7.7583 N1.3577 O1.4773 S0.0417, and these values

can be modified to change the elemental composition used for the averagine. The values of the

averagine and the M/Z of the most abundant peak in a cluster are then multiplied together to

generate the theoretical profile. The default setting is most useful when dealing with an

unknown spectrum.

Averagine Tolerance – default is 10 in a unit of ppm. This value sets the threshold for which a

theoretical isotopomer in a theoretical peak cluster can be matched to an experimental

isotopomer in an experimental peak cluster, where a lower tolerance demands that the theoretical

and experimental isopotopomers have closer ppm values.

Averagine Tolerance of isotopic mass point =

; the bigger intensity of

observed or theoretical is used in the denominator. It is important to note that modifying this

value does not affect the fit score that is generated.

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Move Mode – default is true. Setting to false prevents the user from shifting along the mass

spectrum with the left and right arrow keys in the toolbar of the Spectrum View window.

Parameters – Miscellaneous

Figure 22. Miscellaneous parameters in the Parameters Window

Show Fragment Labels – default is false. Setting to true shows the fragment Input Chart labels.

Show input spectrum – default is true. Setting to false removes the spectrum that was

generated in the Spectrum View window from the user‟s view.

Parameters – Peak Parameters

Figure 23. Peak parameters in the Parameters Window

Signal to Noise Threshold – default is 3. Noise is determined by the minima of the most

abundant isotopomer, and the signal over noise (S/N) parameter defines the “smallest” peak at

the lowest S/N to be picked (Refer to Page 7).

Draw Fitting – default is false. Setting to true draws a curved line that fits the theoretical peak

cluster of the isotopomers. The fitting is visible only when the fitting curve color is not

transparent.

Fitting Curve Color – default is transparent. This can be changed to a variety of colors.

Minimum Background Intensity – used for calculating the level of noise.

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Parameters – Quantitative Analysis

Figure 24. Quantitative Analysis parameter in the Parameter window

Number of Isotopic Points – default is five, which means the top 5 most intense peak within a

peak cluster will be summed to generate the quantitation score.

Analysis Mode – default is line, which means the calculation is summing the absolute intensity

of the isotopomers selected. This can be changed to area.

Parameters – Sequence Table

Figure 25. Sequence Table parameters in the parameter window

Row Height value – default is 32, and changes the height of each row in the grid of the

Sequence Table.

Column width value – default is 32, and changes the width of each row in the grid of the

Sequence Table.

Number of columns – default is 20, and reformats the grid of the Sequence Table, adding or

removing more columns.

Select font – default is Arial, 14pt, and changes the font of the amino acid letters in the Sequence

Table.

Coverage Color – in the middle-down or bottom-up mode, this shows the coverage of the amino

acid sequence that can be identified from the spectra.

Highlight Color –in the middle-down or bottom-up mode, this highlights the fragment of the

amino acid sequence that is selected from the Identification List.

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Spectrum View

Figure 26. The Spectrum View window showing an example dataset.

The Spectrum View window contains many functions that help users to visualize their data.

The window is partitioned into three separate tabs (Spectrum, Chromatogram, and Data) (Figure

26), and their features are highlighted in the space below:

Spectrum View – Spectrum

This displays the mass spectrum of the selected experiment file. This window is populated with

spectrum data as soon as the user open a raw data file. The initial view is the entire spectrum,

but users are able to zoom in on particular M/Z regions by clicking and dragging the mouse to

form a box over the peaks of interest.

After extraction, Spectrum View window can also display identified isotopic peak clusters.

Selecting an isotopic peak in the Mass List automatically displays the peak distribution in the

window (Figure 27).

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Figure 27. The isotopic distribution of a selected peak cluster

Spectrum View Graph – Interpretation for the various characteristics of the graph (Figure 27).

The spectrum generated is plotted with absolute Abundance (Intensity) on the y-axis, and

M/Z on the x-axis.

The chemical formula shown above the spectrum is generated from the average

composition of organic elements in amino acids, known as the averagine. The values

used for the averagine can be modified under Mass Parameters in the Parameters

window. The averagine values are used to generate a theoretical isotopic cluster. The

theoretical isotopic cluster is a calculated guess as to what the chemical formula could be,

and requires verification to be conclusively determined.

The red circles are the isotopomers of the theoretical isotopic cluster, and how closely

these isotopomers match the experimental data generates a fit score.

The green circle is the most abundant isotopomer.

The blue circle is the monoisotopic isotopomer.

The yellow circle represents a theoretical isotopomer that is missing from the experimental

data.

Once the data has been analyzed, the user can then highlight specific peak clusters, and observe

Most abundant peak

Monoisotopic peak

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them in the spectrum view. In Figure 28, peak clusters in the Mass List are selected and

highlighted with purple diamonds.

Figure 28. The Spectrum window showing the selected peaks from the Mass List

By clicking and dragging on the Spectrum View, users can then view particular peak clusters, or

groups of peak clusters more closely.

Spectrum View Toolbar – The toolbar (Figure 29) located at the top of the Spectrum View

window provides several functions to help users examine the mass spectrum.

Figure 29. The toolbar of the Spectrum View window

Shifts the view of the m/z spectrum either to the left or the right. The amount of

adjustment that is made is relative to how much the user has zoomed in.

The „+‟ and „–‟ magnifying glasses zoom in or out of the mass spectrum respectively.

The magnifying glass with the green arrows resets the zoom to the original view.

Changes the graph to either represent the Spectrum, Chromatogram, or Data

tab, where Distribution goes to the Data tab.

Selects the scan that you want to display. If there is only one available scan, the

user will be unable to change this number.

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Deconvolutes the mass spectrum data, generates theoretical profiles and matches these

to experimental profiles. Depending on the size of data file as well as the defined parameters,

this process may take a few minutes. To view the progress of the extraction, there is a bar at the

lower right hand corner of the screen that fills up, and text at the bottom left of the screen that

says how many spectral peaks there are as well as how many have been analyzed.

Performed after extraction, this is used to append any changes made in the parameters

to your spectrum.

Shifts the theoretical spectrum either to the left or right by one dalton. This can be used

to match the theoretical peak more closely to the experimental peak. Doing so can change the fit

score of the match (Figure 30).

Figure 30. Dalton Shift: Move the theoretical peak clusters left or right can adjust the fit score

Either increases or decreases a given theoretical peak cluster by 5%. This can be used to

40% 83%

100% 73%

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match the theoretical peak cluster more closely to the experimental peak cluster.

Perform a new scan on a selected m/z region to search for peaks. The parameters of

the new search can be modified to help identify peak clusters that would not have been identified

under the original parameters (Figure 31 & 32).

Zoom in

Select M/Z range

Change S/N Threshold

Press “Start”

Make sure that a peak in the range of interest is selected before performing the “New Match” function

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Figure 31. Elaboration of the New Match function

After the data processing is finished, the notification is shown at the bottom left corner of the

window.

New identified masses are shown at the end of the list

Figure 32. Results of performing the New Match function

Spectrum View– Chromatogram

This displays the chromatogram that was generated for the experiment if it was generated. This

is a measurement of the TIC (Total Ion Current) intensity as a function of the retention time

(minutes).

New masses ranging

within m/z 1385-1485

Before After

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Spectrum View– Data

This displays a table containing the following fields:

- # – displays the number of the peak in the peak cluster, assigning each peak incrementally

from left to right, starting at one.

- M/Z Theoretical – gives the M/Z value of the theoretical isotopomer to four decimal

places.

- M/Z Experimental – gives the M/Z value of the experimental isotopomer to four decimal

places.

- Intensity Experimental – gives the absolute abundance value for that selected isotopomer

in the peak cluster.

- Relative (Exp) – gives the abundance level of each experimental isotopomer relative to the

most abundant experimental isotopomer, where the most abundant experimental peak is 100.

- Relative (Theo) – gives the abundance level of each theoretical isotopomer relative to the

most abundant theoretical isotopomer, where the most abundant theoretical peak is 100.

- Notes – mentions if the peak is the monoisotopic peak, the most abundant peak, and the

averagine mass point difference in ppm between each theoretical and experimental peak in

the peak cluster.

The Data fields for a given data file initially blank upon opening a data file, and are

populated with information following mass deconvolution when a peak cluster has been

identified.

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Experiment Window The Experiment Window presents information concerning the status of any experiments in a list

representation. This window contains three individual columns (Figure 33): Experiment, which

gives the name of the experiment, Mode, which gives the mode that the experiment is being run

in (ECD, CAD or Full) and then information about the current experimental progress.

Figure 33. Experiment Window

The experiment that is currently being viewed is shown as the checked box. Multiple boxes can

be checked, and the user has control over how they would like these experimental windows to be

viewed.

Handling Multiple Experiment Windows –

An Experiment Window can be created for each file that is opened. This is done by opening

multiple files and then selecting a layout (e.g. Cascade, Tile Vertical, Tile Horizontal) under

Experiments in the main toolbar.

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Figure 34. An example the Tile Verticle layout for multiple experiments

When multiple Experiment Windows are open, several changes are made:

First, the title of each Experiment Window becomes the file pathway of that data file when

multiple experiments are shown for clarification (Figure 34).

Second, the Experiment Windows become selected by clicking and dragging the title bar,

and can be resized by clicking and dragging on the edges.

Third, users have the options to minimize, maximize and close an Experiment Window.

Maximizing a window makes the selected Experiment Window appear the same as a

layout that contains only a single Experiment Window, removing the other Experiment

Windows from view, and removing the file path from the title. When closing an

Experiment Window, the user will be prompted with a message confirming that they

want to close the window, and the window cannot be opened again.

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Sequence Table / Theoretical Data

Sequence Table

The sequence table shows a sequence of amino acids that accompany the series of experiments,

and there are many ways in which this information can be uploaded (Refer to the Workflow and

Parameter section). An example of a fully analyzed sequence table is shown in Figure 35.

Figure 35. An example of the a MS/MS dataset companied with reference amino acid sequence.

Amino Acid Modification – After the sequence data is uploaded, it can be modified to account

for post translational modifications (PTMs). To add modifications, users need to selects the

amino acid that will be modified by clicking the amino acid. The selected amino acid turns bold,

and the title of a box at the bottom of the screen will become updated to X Information, where X

is the type of amino acid that was selected (e.g. Cysteine, Histidine, etc.). Modifications can be

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added from a selected list of known modifications. If the list does not contain a particular

modification, a custom modification should be used when the mass difference is known. To

erase any modifications that have been made to the sequence, select the Clear Assigned

Modifications button at the bottom of the Sequence Table window.

In an MS/MS experiment mode, users can specify whether b and y, or c and z ions are

used. In middle-down experiment mode, they can also provide information about the enzyme

that was used for digestion as well as the number of missed cleavages.

The Tolerance section of the Sequence Table window is used to adjust the error range for a

fragment to be considered a match with the experimental data. This tolerance information can be

set in either daltons or ppm.

The Sequence Table shows different information for different experimental modes. In top-

down full MS/MS mode, the b-y and c-z ion pairs resulting from fragmentation can be shown

using cleavage indicators (Figure 35). In top-down full MS mode, no fragment ion type will be

displayed. In middle-down mode, peptides resulting from enzymatic digestion according to the

enzyme selected will be generated and matched to peak clusters in the Mass List. If the match is

within the tolerance, the sequence will be marked with color in the Sequence Table. Selecting a

certain peak cluster from the Mass List will also highlight the matched sequence in the sequence

table (Figure 36).

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Figure 36. The Sequence Table showing the coverage of the fragments from a middle-down experiment.

Organizing Your View

Pinning Windows

Pinning windows is a simple tool that lets you organize your windows. It is particularly

helpful if MASH Suite is run on a laptop or a computer with a small monitor. Pinning a window

minimizes that window, reformats the other windows to take up its space, and creates a tab that

can be selected to maximize the window. To pin a window, click the pin icon located at the

upper right hand corner of the window, located next to the exit icon. To unpin a window, simply

click the pin icon again, and any windows that were reformatted into the window‟s space will go

back to their original positions.

If the pinned window contains multiple tabs (e.g. Sequence Table and Spectrum View) the

user can selectively open the tabs by selecting either the tab that is highlighted (shown in view)

or the adjacent smaller tab, which will open up the tab that is not seen.

Error Handling

Error Message:

“Unable to instantiate Finnigan objects: XrawFile from Xrawfile.dll (version 2.0.0). Please

check that the following dlls from Xcalibur are available on your system: CFRDBResources.dll,

CFRUtil.dll, ExploreDataObjects.dll, ExploreDataObjectsManaged.dll, ExploreDataObjects.dll,

Fcontrol2.dll, Fglobal.dll, Fileio.dll, finDB.dll, finSSClientLib.dll, Fregistry.dll, XrawFile2.dll”

Error Solution:

This error message most likely appears when the user either did not properly install XCalibur or

unzip the files after the download. To unzip the files, find XCalibur 2.0.7.1044 in your

Downloads folder, right-click the folder, and select Extract All. Once the files are extracted, run

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Setup.exe inside the folder to run the XCalibur program. This will download the necessary .dll

files. Though it is necessary to restart your computer to run XCalibur, this is not necessary to

run MASH Suite.