MultiNotch MS3 Quantitation Solution Developed for Proteome Sciences

The PEAKS development team has developed a solution to address MultiNotch MS3 Quantitation, currently in use by Proteome Sciences for biomarker discovery. MS3 provides more accurate quantification for isobaric tagging experiments, specifically improving TMT and iTRAQ based studies.

In 2011, Gygi et al. reported isobaric labeling mass spectrometry–based proteome quantification commonly suffers ratio distortion owing to protein quantification interference.¹ A more recent publication by the authors identifies that the combination of quantitative isobaric reagents and the MultiNotch MS3 method improves the dynamic range of reporter ion quantitation, reducing reporter ion signal variance, and ultimately produces more high-quality quantitative measurement.² Our development team implemented a data analysis solution to address these points in order to generate more sensitive and accurate results for end users.

The MultiNotch MS3 quantitation solution is currently in use by Proteome Sciences.

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New Methods of Peptide Footprinting

Earlier this year Methods published an article entitled Using Hydroxyl Radical Footprinting to Explore the Free Energy Landscape of Protein Folding. The article provides guidelines to ensure best practises when protein mapping using the emerging technology called fast photochemical oxidation of proteins (FPOP). Recently, BSI spoke with lead author, Dr. Antonio Calabrese from the University of Leeds to discover more about his research. Understanding structure, flexibility and dynamics of side chains in different protein conformers is critical to structural biologists. While very successful for other applications, nuclear magnetic resonance (NMR) is limited in its ability to perform direct comparison on partially folded proteins due to conformational exchange; thus other methods are required.

Dr. Antonio Calabrese, University of Leeds

FPOP is a novel mass spectrometry based method for protein footprinting that has the ability to characterize protein variants in different conformational states. This is achieved by determining conformational changes in proteins caused by folding/ unfolding, with the added capability of investigating changes in solvent accessibility that accompany ligand binding. Further, the speed of FPOP avoids conformational averaging. According to Dr. Calabrese:

The lab is very interested in method development, developing new techniques and applying a variety of mass spectrometry technique to study protein structure. We also work with native mass spectrometry and chemical cross-linking and so… labeling with hydroxyl radicals is complementary to hydrogen deuterium exchange (HDX). One of the things different about it is because you have a covalent label you can be a bit more rigorous with your downstream sample handlings.

Within the realm of protein mapping, a significant interest of the group is surface mapping. To accomplish this, the surface area of a protein is labelled with hydroxyl radicals; opening the opportunity to identify labelling sites at the amino acid level and perform quantification on the observed oxidation.

Dr. Calabrese was not shy about addressing the challenges of FPOP: The thing that is rather complex about the data that you get out of FPOP is that generally speaking you are looking at modifications which are quite lowly abundant and so being able to reliably quantify those levels of oxidation and quantify those modifications is often quite challenging; especially if you want to analyze your data in order to obtain very specific information…. We used PEAKS to help assign our modification sites and then we also had to manually validate, specifically the modification, sites.

Peptide level quantification of oxidation levels.

In addition to using PEAKS for identification, the quantification node, PEAKS Q, was applied in both label and label free techniques. Precursor Ion Quantification (traditionally used for ICAT/SILAC) was configured for custom variable modification mining and Label Free Quantification for the remainder of features. When asked about satisfaction from the automated results produced by PEAKS the answer was very positive:

The automated results were definitely quite good. Whenever we manually validated those automated results they were pretty much spot on. When results are right it is obviously much easier to work with. Also, the capacity to look at the de novo spectra means you have got a lot more data to play with; otherwise much of it just gets thrown away. […] Now we are using it routinely for all of our DDA data analysis in the lab.

As with many other emerging technologies, the project is set to progress onto other complicated systems where it can continue to be complementary to HDX.

We have just looked at some relatively simple systems [soluble proteins], but now we are starting to look at more complicated systems using the techniques; so now are starting to look at characterizing membrane proteins which are obviously a lot more challenging and trying to optimize everything now.

We are grateful to have had the opportunity to speak with Dr. Antonio Calabrese and receive insight into emerging methods that dig deeper into explaining our proteomic world.

For those interested in learning more about the PEAKS software, regularly scheduled public webinars are available for free with our support team here.

PEAKS Training Workshop: Stanford, California

The PEAKS Training Workshop is coming to Stanford University (Stanford, California) on August 24-25.

The Training Workshop provides a full hands-on experience with PEAKS 7.5 for both new and experienced users of PEAKS Studio. In addition to demonstrating the logic and algorithm behind PEAKS software, the workshop focuses on how PEAKS may be used specifically toward the users’ research and applications.

Participants of the course can expect to increase their knowledge of LC-MS/MS data analysis and result validation upon completion of the course. Visit our training website for more details and to sign up.

PEAKS Studio 7.5

We are excited to announce the release of PEAKS Studio 7.5. There are many new features in this release that expand the capabilities for our users. Just to name a few favourites, consider:

• PEAKS PTM Profiling – to detect and quantify modifications
• Peptide Mapping - a powerful comparison tool, providing an intensity map of all the peptides associated with any particular protein.
• Peptide Feature Intensity – in one click users can analyze and quantitative peptides across all samples.
• Automated Multi-Round Search – automatically filter out high quality spectra unmatched after database searching perform additional searches against different sequence databases or change parameter settings to help explain more from your data.

These were designed to further compliment PEAKS’ proven track record of high accuracy and high sensitivity in third party studies. A complete list of improvements and additions to the software can be found here: PEAKS Studio 7.5.

ASMS 2015

The expanding popularity of PEAKS made for another exciting ASMS annual conference. During the PEAKS Meeting on Sunday, BSI gave an early glimpse into a future version of PEAKS Studio. Guest speakers also presented current research projects where PEAKS proved vital:

• Jeanne Rumsey of Washington University in St. Louis,
• Database Search Strategies for Biomarker Design in Infectious Diseases

• Dr. Cheryl Lichti, University of Texas Medical Branch,
• PEAKS and the Quest for Single Amino Acid Variants

During the conference a number of notable oral presentations and posters resonated well with attendees and emerging methods. (Please note you will need to login using your ASMS details to view the presentations.)

• Ming Yao, Bristol-Myers Squibb
• Untargeted and Rapid Detection and Characterization of Modified Monoclonal Antibodies using LC-TripleTOF and Multivariate Statistical Analysis,(Slides)

• Josh Elias, Stanford University
• Fast and Accurate Unrestricted Spectrum Interpretation: You Don’t Know What You’re Missing.

A significant list of oral presentations and posters relevant to PEAKS users are available on our ASMS 2015 webpage. Our own poster, Peptide Mapping for Quantitative Profiling of Peptide Variants using PEAKS is also available.

ABRF iPRG 2015

The purpose of this year’s ABRF iPRG study was to evaluate different data analysis methods for relative protein-level quantification in label-free proteomics. In conclusion of the study, PEAKS label-free quantification proved to successfully demonstrate a high-level of accuracy in determining relative protein abundance.

 

Graphs from the results were retrieved from the ABRF iPRG Preliminary Results Summary. To view the full Preliminary Results Summary, please use the link provided below.

 

The first graph shown is an overview of all the participating Study IDs in relation to their results as seen on slide 20 in the Preliminary Results Summary. The same graph is then presented again on slides 24 and 25 (zoomed in), to show the specific tools used for peptide ID, as well as quantification for each submission. .

 

Of the 51 submissions, very few were able to recognize the true differentially abundant (DA) proteins, as seen in the graph. PEAKS, however, recognized all 6 true DA proteins (represented in blue), and reported 0 false DA proteins (represented in red).

 

 

http://www.abrf.org/ResearchGroups/ProteomicsInformaticsResearchGroup/Studies/vitek-iPRG15.pdf

 

 

Participating researchers were required to detect and identify proteins that changed in relative abundance among four biological samples, prepared from the same S. cerevisae yeast cell lysate.

 

To truly focus on the statistical assessment of the varying data processing approaches, the peptide identifications and their associated integrated peak intensities were provided to all participants.

PEAKS in Germany: Proteomic Forum 2015

Come by and visit PEAKS at this year's DGPF: Proteomic Forum (booth 19). The conference will be taking place March 22-25 at the Technical University Berlin (Germany).

In addition, PEAKS will be hosting a Training Workshop, March 19-20, 2015, at the Crowne Plaza, Berlin City Center. The workshop is designed to give users a hands-on approach to fully understanding the capability of PEAKS. Visit http://www.bioinfor.com/ for more information and registration.

“The German Society of Proteomic Research coordinates and provides a network comprising academic institutions, commercial companies and special institutions at the national and international level in order to support the extension of scientific knowledge in the field of proteome research and protein analysis.”

For more information, visit http://www.proteomic-forum.de/

PEAKS at US HUPO 2015

Stop by Booth 11 to visit PEAKS at the 11th annual US HUPO Conference. This year’s conference, located in Tempe, AZ, will span three days and include the research poster “Quantitative Analysis of Native Peptides with LC-MS/MS Directly from Complex Secretome Samples” in Computational Methods and Statistics.

From live demos to direct support, we want to show you why PEAKS is right for your research.

“US HUPO is a non-profit educational and networking association for those engaged in proteomics technologies and research pertaining to the human proteome and model organisms.”

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.

Merry Christmas and Happy New Year!

2013 will come to the end in a few days. It's been a great year for us, the PEAKS team, at BSI and we want to take this opportunity to wish everyone a Merry Christmas and a Happy New Year!

Navigate through your LCMS data in PEAKS

PEAKS 7 is released with a re-engineered LCMS view. Users may use this integrated and interactive view to navigate and dig into the data and result easily. In this post, I am going to talk about the controls and functionalities we have built into this view.

The LCMS view can be found on any opened data nodes, identification results and label free quantification results. It contains four main components: the main heatmap view, the control panel, the TIC and the thumbnails.

Heatmap View

In the heatmap, we use the color to represent the strength of the LC-MS signals. The darker the color the stronger the signal. The x-axis is the m/z and the y-axis is the retention time.

Navigation control

The operations to zoom in/zoom out and move the view is very similar to Google Map. Scroll the mouse wheel forward to zoom in and scroll backward to zoom out. Drag the mouse while holding the left mouse button to move the view. In some situation, you may want to zoom only on one axis. This can be achieved by scrolling the mouse wheel while the mouse cursor is on the RT axis or the m/z axis. You can also drag the mouse holding the right mouse button to quickly zoom into that selected area if you know exactly where you want to look at.

Adjust the contrast

Scrolling the mouse wheel while holding the CTRL key will adjust the contrast of the heatmap. This is particularly useful when looking at less intense area that the default contrast cannot visually distinguish the signal strength. Here is an example. The heatmap is zoomed into a very small region. The image on the left uses the contrast works better when zooming all the way out and viewing the entire heatmap. But in this local region, the colors are all the same. You cannot visually distinguish the strength of the signal. The image on the right displays the same region with the contrast adjusted.

Control Panel

The control panel is on the upper right corner of the heatmap view. It provides information such as the m/z and RT of the mouse cursor location, along with many other functions associated with the heatmap.

Intensity display cut-off

The slider (0) can be used to adjust the intensity cut-off. Any peaks with an intensity less than this cut-off will not be displayed. This is very useful when you want to focus on the real signal and get a much cleaner view. This slider only affect the heatmap display. We use a much sophisticate algorithm to do the noise removal in our feature detection.

The Buttons

The back and forward buttons (1) provide an easy way to restore the view to a previous state. PEAKS remembers all the operations you have done to the view, including zooming, moving and changing contrast.

The 1:1 button (2) will immediately zoom out in the view to display the full heatmap. You can also invoke this function by double clicking your left mouse button anywhere in the heatmap.

The 3D button (3) toggles the view between heatmap and 3D mode. While the heatmap mode is sufficient in most cases, 3D mode may provide a direct visualization on the signal intensity in some situations. Below is an example displaying the same data in different modes. While in the 3D mode, most of the navigation controls are the same as the ones mentioned previously, there is one minor difference. You are not able to change the contrast in 3D mode, instead, holding CTRL key while scrolling the mouse wheel will change the height of the peaks.

The export button (4) is self-explanatory. Many images in this post are created this way. It supports over-sampling up to 8x, which will generate a much bigger image with smooth font for print.

The fold button (5) toggles the control panel between the full panel mode and mini panel mode. The mini panel can save you some screen space especially on old, low resolution monitors.

The synchronize button (6) will synchronize the m/z and RT position across all samples in the result. This is very useful when you are examining a peptide feature and wondering what the same area will look like in other samples. Instead of manually navigate to the same m/z and RT region in a second sample, you only need to make sure that this button is selected and then change to the sample you want to look at. PEAKS will automatically navigate to the same area with the same zoom level and contrast.

The help button (7) displays a small dialog to provide a quick help on the navigation controls.

Jump to an area quickly

The text box (8) can be used to jump to a m/z and RT position quickly if know the numbers. The format is m/z white space RT (e.g. 420.83 26.25). PEAKS will center the view on the provide coordinates and determine an appropriate zoom level.

Display options

In the control panel, there is a group of checkboxes (9) that controls whether or not to overlay certain information on the heatmap. Depending on the result type, it may include features, MS/MS spectra, identified peptides and de novo only tags.

The following example is from a PEAKS DB result with all the checkboxes selected. The pink circle represents the center of a peptide feature. The empty blue square indicates a MS/MS spectra. The solid blue square means that the MS/MS spectra has a confident peptide identification passed the score filter. The solid yellow square means that although the MS/MS spectra does not have a confident identification, it produced a de novo only tag.

Mouse over the circles and squares will display more information. Clicking on the squares will pop up the spectrum alignment view and you can examine the MS/MS spectra directly.

In the label free quantification result, you may see two type of pink circles in the heatmap view, solid and empty. They all mean that PEAKS has detected a feature there, the solid circle indicates that the feature has passed the filters. Clicking on a pink circle will highlight the approximate boundary of that feature. You can highlight multiple features by holding the CTRL key when clicking the mouse button.

TIC

PEAKS displays the TIC beside the heatmap RT axis. You can zoom in/out on the RT axis by scroll the mouse wheel on the TIC. When zoomed in, PEAKS will highlight the chunk of RT that is being displayed in the heatmap view. You can drag the boundary of the highlighted portion to adjust the zoom level or you can drag the middle of the highlighted portion of move the heatmap content along the RT axis.

Thumbnails

The thumbnails are small, un-zoomable version of the heatmaps. It provides an overall preview of the sample. The contrast is connected to the corresponding heatmap, so changes to the contrast of a heatmap also apply to the thumbnail. PEAKS displays a rectangle on the thumbnail to highlight the area current viewed in the heatmap. You can adjust the area by scrolling the mouse wheel on the thumbnail or drag the borders, effectively, it zooms in/out in the heatmap. You can also drag the middle of the rectangle to quickly move the viewing area in the heatmap.

If the result related to multiple samples, all the thumbnails will be listed here. You can quickly switch between samples by a simple clicking on the sample thumbnail you want to look at.