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Frequently asked questions about ProTrawler

Questions

Answers

Support

What file formats does ProTrawler support?

See our list Supported Raw Data Formats.

Does ProTrawler support profile and/or centroid chromatograms?

ProTrawler6 supports profile chromatograms. ProTrawler7 will support both profile and centroid chromatograms.

Installation

How do I install Enterprise-licensed ProTrawler?

Double click on the installer. Read the EULA. If you accept the terms, follow the installer's instructions. Enterprise-licensed ProTrawler may be installed anywhere on your hard drives.

How do I install ProNets-licensed ProTrawler?

You must have write permission on the C: drive so that c:\bainc is created during the installation process.

Double click on the installer. Read the EULA. If you accept the terms, follow the installer's instructions. ProNets-licensed ProTrawler may be installed anywhere on your hard drives.

Is there a User Guide for ProTrawler?

Currently, a User Guide is shipped with the ProTrawler installer. To access this user guide, select Help->ProTrawler Tutorial from the menu bar.

Validation

How do I validate ProTrawler?

The best way to validate ProTrawler is to compare individual and co-added scan values with what the instrument vendor software reports. ProTrawler should be exactly the same in both m/z and intensity. If not, please report it.

You may also wish to compare deconvolved spectra from ProTrawler and the-software-that-came-with-your-instrument. There may be disagreements, but we are more likely to defend our results than attempt to reproduce whatever your vendor's software gives.

What is the limit on file size?

As of version 6.1.0928, we read flies up to 2^31 = 2147483648 ~ 2.1GB. This has been large enough for every file we've seen in two years of version 6, except for one.

How can I keep the Windows task bar from overwriting the bottom of the ProTrawler window, obscuring the mass scale of the reconstructed ion plot?

You can drag the Windows bar to the bottom of the screen, or you can change the shape of the ProTrawler window by hand.

How do I adjust the horizontal axis in both directions?

The control buttons on the left side of the display panels can be used for zooming. The big plus sign with arrows is the zoom-to-fill feature. There is a toggle button for mouse zooming. In one state, it auto-zooms the y axis to the base peak in the display. In the other state, it requires you to click-and-drag and draw a box for display. Between these two, you should be able to see the desired portion of the scan.

One needs the ability to either freely move peak labels or remove the non-peak label text that is put onto the spectra (sample info from MassLynx file, I think). How do I keep the sample info from ending up on top of an important peak label?

Yes, this is a good usability comment. It is being fixed in version 7. For the present, please use the zoom creatively to make sure that the labels don't overwrite each other.

How do I label peaks?

ProTrawler uses two parameters for labeling apexes (not actual peak centroids.) They can be found in the MenuBar->Preferences. One is the number of peaks to be labeled. The other is the effective resolution of the labeling (which is mislabeled in the popup box.) If you want a resolving power of 10000 in your peak labeling, viz., the ability to label apexes at 10,000 and 9999, then set the resolution to 0.00001.

It would be nice to be able to print out 2 windows at the same time (e.g. reconstructed mass and scan data or reconstructed mass and TIC data)

We provide the ability to copy these figures to a clipboard for reassembly in PowerPoint or whatnot. Then you can display/print them side by side.

How do I delete a scan?

In the m/z scan display panel (the middle panel), right click on the scan name and select delete.

Usage

Viewing the chromatogram

Can I see an extracted ion chromatogram for reconstructed mass (i.e. show me the intensity of insulin (mass=5734) as a function of time in this HPLC trace)

Not presently. This is a Feature Request. We would show the extract ion chromatograms for all detected charge states of the zero-charged mass.

Developing a Model

Background Subtaction
The backgrounding step does not seem to remove much from the file. I know it is not necessarily supposed to, but there is a chemical noise background (a few counts of intensity across the entire spectrum) that I personally do not feel is significant. Is there something that can be done with the degree of fit?

First, use the Primary and Secondary filtering in the Peak Shape Deconvolution widget. Set them high, 99% and 3. If you set them to 0% and 0, then *all possible* features are preserved, regardless of statistical significance.

Second, you may set the degree of fit to 2 in the background subtraction widget to raise the background estimation to its maximum. Use

  • 0 for clean samples and peptides
  • 1 for proteins
  • 2 for protein mixtures and messy samples
What's a good way to quickly ascertain the width of a feature for background analysis in ProTrawler without having to go into <<instrument vendor software>> and count points?

Just eyeball it and estimate "10" when the number is obviously between 5 and 15 or "50" whether the number is obviously between 25 and 75. This is NOT a restrictive parameter.

Peak Shape Modeling
Peak Shape Deconvolution
Peak Filtering
How can I make the filter more selective?

Set to higher values. 95% and 2 or 99% and 3. Remember that errant peaks that do not line up in charge state series or isotope series are ignored anyway.

Zero-charge Mass Reconstruction
Can we display the zero-charge mass in "profile" mode?

You'd like to draw a Gaussian at the deconvolved mass with the width given by the estimated error? It is easy to draw the Gaussian, but it is a conceptual step backwards in the ProTrawler-Regatta work flow. We seek to reduce continuum data to list data, then handle list data in an internally-consistent fashion using Regatta's fairly-sophisticated list handling based on our solution of the problem posed by fuzzy lists using our implementation of the Transitive property of equality. [If A=B and B=C then perforce A=C. This statement must be true for arithmetic to be defined on a set. When this statement fails, as in the case of poorly described spectral data, then further operations develop artifacts, such as order dependence.]

What can I do to get rid of the smaller m/z peaks to simplify mass reconstruction? The vast majority of my customers are interested in the significant (>5%) components for most of their analyses.

The best way to handle this is through filtering the peak list after shape deconvolution. Alas, there are also many people who are specifically NOT interested in the biggest peaks but want to do discovery on the smallest ones.

Would it be possible to have ProTrawler go back and look for all possible charge states of a found protein mass in the scan data? This is a way to address the problem that arises when some of the charge states are a slight bit outside the mass error window and get missed in the “visible” display.

Use the m/z tolerance feature in the zero charge deconvolution. Set it from .1 to .2 or higher. That will pick up those m/z's that are slightly off position.

Deisotoping
Why would one not select the z=0 button?

Lots of people are happy with m/z deconvolutions to monoisotopic m/z rather than zero charge monoisotopic mass. Perhaps a popular ms/ms search engine doesn't require zero-charge masses because that's "too hard" on the existing instrument vendor software!?!?! A good reason to use ProTrawler!

When I deisotope, sometimes I get two peaks reported back with nearly identical masses (e.g. 3191.2 and 3191.18 or 3299.71 and 3299.78). These should have been interpreted as the same peak (mass error set to 0.1 Da)?

No, there can be nearly degenerate, overlapping isotopic states. ProTrawler will tease them apart based on shape deconvolution. Also, for isotopes, your m/z errors are usually a low lower than 0.1 m/z. Perhaps you could go to 0.05 m/z or lower.

How wide is a feature when looking at an isotope pattern for background subtraction?

The [Acronyms/FWHM|FWHM] width, in data indices, of one isotopic peak. It is usually 5-10 indices.

How do I use the "cluster" feature with deisotoping?

To use the cluster feature with deisotoping (which is recommended) you should select the cluster radio button, then select the whole cluster with the mouse (click to the lift, click to the right) but enter the width of a single isotopic peak. ProTrawler will do a best-fit of a peak shape to *all* isotopes in the cluster.

Be sure not to select a cluster with obvious monoistopic mass degeneracies!

Also, how are the intensities for the isotopically-resolved peptides determined?

ProTrawler adds the detected signal "under the curve" for each isotope and for each charge state (if you've selected the z=0 option.) When two peptides have nearly the same mass, the numerical engine breaks the signal into proper proportions.

Known Issues

Updates, upgrades and reinstallation

The installer for ProTrawler or ProAngler (like Regatta's installer) requires full uninstallation of the existing version before it can be upgraded.

View/Model tab disappearing data

From time to time, ProTrawler has been known to drop pointers on the View/Model tab. This does not cause a crash. If you delete the non-responsive scans by hand, ProTrawler will continue to function without problems. -PEL 7/31/2007

Preferences and Peak Labeling

The label "percent" should read "Resolution". It describes the exclusion range over which a higher apex will exclude the labeling of a lower apex. This will be changed shortly to "Resolving Power".

Display of specific isotopes

Selecting zero-charge masses after deisotoping does not highlight the m/z peaks used in the deisotoping process.

Absolute file size

As of version 6.1.0928, ProTrawler can read individual files up two 2^31=2147483648 bytes ~ 2.1 GB. We can read "chromatograms larger than that, but the chromatogram must be made up of files smaller that that limit. Also, we open and parse files larger than the 2.1 GB limit as long as no reads go to bytes with addresses greater than 2.1 GB. This will be resolved in a bug fix release.

Recent Fixes

The zero charge deconvolution tool was not correctly implementing the minimim and maximum adjacent charge feature. This has been correct in version 6.1.0928.

Licensing

ProNets licenses

What is a ProNet?

A ProNet is a license for for a single chromatogram. It uses a special, yet functionally identical version of ProTrawler that is tied to individual chromatograms.

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