Analysis of histone modifications with PEAKS 7

The complex nature of histone modification patterns has posed as a challenge for bioinformatics analysis over the years. Yuan et al. [1] conducted a study using two datasets from human HeLa histone samples, to benchmark the performance of current proteomic search engines. This article was published in J Proteome Res. 2014 Aug 28 (PubMed), and the data from the two datasets, HCD_Histone and CID_Histone (PXD001118), was made publically available through ProteomeXchange. With this data, the article uses eight different proteomic search engines to compare and evaluate the performance and capability of each. The evaluated search engines in this study are: pFind, Mascot, SEQUEST, ProteinPilot, PEAKS 6, OMSSA, TPP and MaxQuant. 

 

In this study, PEAKS 6 was used to compare the performance capabilities between search engines. However, PEAKS 7, which was released November 2013, is the latest version available of the PEAKS Studio software. PEAKS 7 not only includes better performance than PEAKS 6, but a lot of additional and improved features. Our team has reanalyzed the two datasets HCD_Histone and CID_Histone with PEAKS 7 to update the ID results presented in the publication by Yuan et al.  These updated results showed that instead, it is PEAKS, pFind and Mascot that identify the most confident results.

 

Proportion of Confident IDs

As indicated in the article, the two HeLa histone datasets were examined by each search engine using the same database search parameters. Seven variable modifications of histone were used in the study, and are reiterated in table 1 below.

 

Table 1. Modification parameters for database search

Fixed modification	Propionyl[Peptide N-term]/+56.02
Variable modification	First (un)	Propionyl[K]/+56.026
	Second (ac)	Propionyl[K]/+56.026; Acetyl[K]/+42.011
	Third (me)	Propionyl[K]/+56.026; Methyl_Propionyl[K]/+70.042
	Fourth (di)	Propionyl[K]/+56.026; Dimethyl[K]/+28.031
	Fifth (tr)	Propionyl[K]/+56.026; Trimethyl[K]/+42.047
	Sixth (ph)	Propionyl[K]/+56.026; Phospho[ST]/+79.966
	Seventh (co)	Propionyl[K]/+56.026; Acetyl[K]/+42.011; Methyl_Propionyl[K]/+70.042; Dimethyl[K]/+28.031; Trimethyl[K]/+42.047; Phospho[ST]/+79.966

 

When the data was run with PEAKS 7 also using these same parameters, an updated comparison of the IDs and confident IDs from the article published by Yuan et al. was created, as shown in figure 1. The comparison includes the results produced by the eight different search engines. IDs (shown as solid bars) from each search engine are identifications with an FDR < 1%; whereas confident IDs (shown as striped bars) are the number of IDs from each search engine which are also present in the ‘all_Confident’ group of IDs. The term ‘all_Confident’ was used to indicate IDs that were found by at least two of the eight different search engines.

Figure 1 (a-g). Comparison of the number of IDs and confident IDs of the seven variable modifications produced by the different search engines using HeLa histone HCD and CID data

(a) indicates the number of first (un) modified ID; (b) number of second (ac) modified ID; (c) number of third (me) modified ID; (d) number of fourth (di) modified ID; (e) number of fifth (tr) modified ID; (f) number of sixth (ph) modified ID; and (g) number of seventh (co) modified ID.

By analyzing each of the graphs presented in figure 1, PEAKS 7 produces the most confident results of the search engines evaluated in the study, along with pFind and Mascot. This is true in all cases (un, ac, di, tr, ph, and co; where ph tied with pFind and Mascot, and co tied for first with Mascot) except in the third modification where pFind and Mascot found the most confident result. 

Running Time
 

For this analysis, PEAKS 7 was run on a typical desktop computer with an i7 CPU and 16G RAM.  PEAKS 7 finished each of the first six searches (un, ac, me, di, tr, and ph) around 22 minutes and then 14 minutes for the HCD_Histone and CID_Histone database searches respectively.  Compared to 2h-7h indicated by [1] using PEAKS 6, the speed of PEAKS 7 is much faster. For the seventh search which involved multiple PTMs (co), PEAKS spent 30 minutes, and then 14 minutes performing the database search for HCD_Histone and CID_Histone respectively.

Therefore, the performance time of PEAKS 7 is very comparable to the other search engines as drawn in conclusion from [1] and consistent with the performance capabilities presented in (http://peaksblog.bioinfor.com/2013/12/boost-your-analysis-speed-with-peaks-7.html).

References

1.     Yuan ZF, Lin S, Molden RC, Garcia BA. Evaluation of proteomic search engines for the analysis of histone modifications.  J Proteome Res. 2014 Aug 28. [Epub ahead of print]

 

PEAKS Training and Workshop, September 1-3, 2014 – Paris, France

PEAKS will be in Paris, France hosting both a training course and a workshop starting this September.

A two-day PEAKS training course and a one-day workshop will be held at the Hotel Paris Bastille located at 67 rue de Lyon, Paris France.

The training course is designed to introduce users to the software, and elevate their understanding to become a confident user. Attendees will gain hands-on data analysis experience with PEAKS from the people who develop and support it.

The workshop is designed for participants facing more difficult challenges in their research. This hands-on PEAKS practice will definitely increase your expertise of LC-MS/MS data analysis and quality control.

More details can be found here. You can also register here directly.

BSI Joins Battle to End Cancer in our Lifetime

On the weekend of June 7th and 8th Bioinformatics Solutions Inc, makers of PEAKS software, was happy to sponsor one of its team members Dan Maloney in the Ontario Ride to Conquer Cancer. It was a grueling two day 235 km ride from downtown Toronto to the top of Niagara Falls.   Through personal donations from friends, family, and co-workers, and a $1500 sponsorship from BSI, Dan was able to raise over $2500 to support the Princess Margaret Cancer Foundation.  Over 5000 riders participated in the event.  Together they were able to raise over $20 million to fund cutting edge cancer research.  This amazing achievement is a new fundraising record for the ride and we are happy to have played a part.

The Princess Margaret Hospital's research program is ranked fourth in the world.  That is in terms of the percentage of publications cited in high impact oncology journals.  It is the research center where important discoveries such as Dr. James Till and Dr. Ernest McCulloch’s discovery of stem cells in 1961 were made.  Currently, they are pushing forward to improve the effectiveness of personalized medicine in the fight against cancer.  Also, one of their researchers, Dr. Tak Mak, has recently received Health Canada and FDA approval to go ahead to phase 1 clinical trials for a new cancer drug that will target PLK4, an important enzyme in cancer cell division.  We are happy to support research such as this happening at Princess Margaret to help end a disease that 2 out of 5 Canadians will be diagnosed with in their lifetime.

When asked about the ride Dan Maloney said, "it was a very rewarding experience, but I was overjoyed to cross the finish line.  Thank you very much to everyone who was able to make a donation.  Very special thanks to BSI for sponsoring me.  Without generous donations like the one you made, large fundraising events such as this would not be possible.  Events such as the Ride to Conquer Cancer are crucial if we ever want to see the end of devastating diseases such as cancer which sadly will affect us or someone we love at some point in our lives."

PEAKS Two Day Workshop, June 24 - 25, 2014 - Boston, MA, USA

The PEAKS team will host a two-day workshop at Hilton Boston Back Bay Hotel in Boston. The early bird registration is open until May 31. You can register the workshop here.

Annual PEAKS User Meeting co-located with ASMS 2014

It is the time of the year again to get prepared for the ASMS conference. We are happy to announce that the 8th Annual PEAKS User Meeting will take place on June 15, 2014, co-located with ASMS.

Please join us and register today to reserve your seat. http://www.bioinfor.com/peaks/corp/conferences/peaks-asms-2014.html

This year, the user meeting will focus on PEAKS applications. Both the guest speakers and scientists from the PEAKS team will talk about real research and how PEAKS may help in the process. We have reserved the room for the entire afternoon. So if you want to discuss how PEAKS may help on your research project, you are welcome to talk to our scientists individually after the user meeting.

If you cannot make it to the user meeting, please do visit us at our booth (#42).

See you in Baltimore!

PEAKS 7 Patch Release

While laying down the foundation of the next major upgrade, we are also improving the current version. Just a few weeks ago, the PEAKS team has released a new build of PEAKS 7 to address some issues in the 2013 November release.

If your PEAKS 7 installation works well for you, you do not need to upgrade to the new build. Here are a list of changes in the new build.

• Improved 3rd party exporting to Scaffold and fixed an issue that sometimes the PTM information may be missing

• Fixed an issue that sometimes the proteins reported in mzIdentML does not have supporting peptides

• Fixed a regression bug affecting charge and mass correction on high charge Orbitrap data

• Third party and pepXML export from inChorus results are now disabled. It was unintentionally opened in the initial PEAKS 7 release. In mzIdentML, there are currently no CV term in the standard for PEAKS inChorus results.

• unique peptide filter in Label Free Quantification results now correctly uses the unique peptides instead of the peptide features as the filter

• Fixed an issue in Label Free Quantification p-value calculation. Users do not need to redo the analysis. Re-open the results will have the p-value displayed correctly

• PEAKS now support 64 bit CompassXtract libraries from Bruker

• PEAKS now can read the scan number from MGF files generated by the new Compass tools

• Better handling on MGF/PKL/DTA files with excessive white spaces

• Better handling on importing Mascot results into PEAKS. Solved issues for some custom FASTA header formats

• PEAKS 7 will now less likely to exit shortly after the start up due to the slow Derby initialization on some system. The default timeout has been increased to 30 seconds

• PEAKS project converter. Now the MS1 spectrum information is correctly converted from PEAKS 6 projects

If you want to upgrade to the new build, please contact sales@bioinfor.com or your sales representative so that we will adjust your license and send you the instructions.

Using PEAKS via Windows Remote Desktop Connection

Recently some users reported an graphics issue when using PEAKS over Windows remote desktop connection. PEAKS GUI is not drawn correctly on the screen.

We have observed the same issue in the team internally and investigations lead us to believe the issue resides in Java Windows look and feel. I did some internal testing and it seems using the cross-platform look and feel solved the problem.

To make things easier for you, here is a modified jar file you can use to replace peaksstudio.jar in the PEAKS directory. Your PEAKS build number (go to Help -> About PEAKS to check) must match the build number of the jar, e.g. build 20131119. Please make sure you backup your original peaksstudio.jar just in case.

Create complex projects with ease in PEAKS 7

One of the issues in previous version of PEAKS is the difficulty on creating large, multiple sample projects. Users have to make hundreds of mouse clicks to create a project with hundreds of samples.

Introduced in PEAKS 7, the new project wizard has all the functionality in the previous versions and provides additional controls to make creating complex project easily. You can still use the new project wizard the exact same way as it is in the previous version to quickly create simple projects containing only a few fractions and samples.

Below is a screenshot of the new project wizard interface. The portion in the red rectangle is the new controls introduced in PEAKS 7. It essentially provides a staging area for all related data files, a search function to help quickly highlighting multiple files and control buttons to add the highlighted files to the project in different ways.

You can use the "Add Data" button to add data files to the staging area. The files will be listed in the white box on the left. Once the files are listed there, you can then type a few characters in the search box to highlight files of interest. PEAKS will gray out the files that do not match the search and pull all the matched files to the beginning of the list.

Once you have one or multiple files selected in the staging area, you can use one of the three buttons to add them into the project.
The first button will create a new sample and put all the selected files under the new sample as fractions. This is equivalent to adding multiple files to one sample in the old way. The second button  will create a new sample for each selected files. This is very useful when creating a PEAKS project with hundreds of samples. The third button is to be used to add more fractions into an existing sample. The forth button is to be used to remove samples and/or fractions from the project.

The new project wizard also integrated the most commonly used analytical workflow in PEAKS, starting from data refinement (data pre-processing) to peptide/protein identification, including PTM analysis and mutations. The workflow may include label free quantification if the context is applicable.

PEAKS 7 Viewer on Mac OS X Mavericks

I wrote a post in early 2013 here to help users to run PEAKS as a Viewer on Mac OS or Linux. Similar procedure applies to PEAKS 7 as well. Many thanks to user ceinwyn that brought the issue to me that PEAKS DB search results cannot be opened.

I spent some spare time looking into this issue and luckily found a workaround. Just want to re-state my disclaimer here.

Disclaimer: PEAKS does not officially support any OS other than Windows as of the time I am writing the post. The software may not be fully functional. Activating the software on OS X or Linux will consume the license, which means the same license can not be used again. I strongly recommend only following the steps to configure PEAKS Viewer (the unlicensed Studio) on OS X or Linux for PEAKS result sharing and presentation purposes.

Basically what happened here is that PEAKS 7 has some sizing issue with the Auqa look and feel from Apple Mac OS X. The workaround is to change the look and feel to a cross-platform one that Apple also support. To make things easier, I have put a modified version of the jar file here. And here are the steps:

1. Check the version of PEAKS. Go to Help -> About PEAKS. In the dialog, you should see the build number, e.g. build 20131119. This build number must match the number in the downloaded jar file in the above link.
2. Close PEAKS.
3. Download the jar file and replace the peaksstudio.jar in the PEAKS directory with this one.
4. Start PEAKS and now the PEAKS DB search results can be opened. 

Enjoy PEAKS and happy sharing!

De Novo Sequencing using Spectral Pairs or Triplets

PEAKS supports de novo sequencing using spectral pairs or triplets that are generated using different fragmentations. This is a feature introduced in PEAKS 7. Here is the poster that briefly described the method.

To use this function, simply specify the fragmentation mode as 'Mixed' during project creation, PEAKS will then automatically detect spectral pairs or triplets when doing de novo sequencing.

There are a few things to watch out though. When the fragmentation mode is set to 'Mixed', PEAKS will try to get this information from the data for each individual spectra. From the many datasets we have tested, this is generally not a problem for Thermo raw files. Other formats, especially some in-house generated XML files(e.g. mzXML, mzML) may be problematic as they either do not include the fragmentation mode information at all or PEAKS won't be able to retrieve the information for various reasons. In this case, PEAKS will ignore those spectrum, thus generating empty de novo sequencing results.

When the fragmentation mode information is available, the spectral pairs or triplets will be formed if the spectrum are from the same precursor ion in the same MS scan. The de novo sequencing result from a pair or triplet will be presented in a single row in the peptide table. The spectrum annotations for different fragmentation mode will be stacked vertically on the screen.

If you click the 'All candidates' button, all the calculated de novo sequences will be displayed in a pop up window. You can then click on those sequences to examine the spectrum annotation from individual fragmentation mode.

In the above example, although PEAKS has figured out the correct sequence using any one of the fragmentation mode, the confidence of the sequence from the triplet is much better.