PEAKS - FAQ

"Thanks a lot, I more and more like the power of PEAKS, it is simply great."

Manfred Raida,
Biomedical Sciences Institutes

FAQ

Here is a collection of frequently asked questions, categorized by application. In addition to this list, using the search field (located in the header section above) may help resolve questions. If you do not see what you are looking for, by all means, Contact Us.

Installation/Configuration

1. Question:How do I register PEAKS Studio?

Answer. The first time PEAKS is run, the license wizard will appear automatically.

Select the first option, request license file (has internet connection). Click "Next".
Enter the necessary registration information. The email address entered here will be used to send you a license file, different from a registration key. If you have purchased PEAKS and have a registration key, looks like PS*************, enter/paste this into the respective field, careful not to include any spaces. For those evaluating the software, request a license file by entering your institution's name in the corresponding lower field. Click "Next".
An automated BSI service will generate the license file (license.lcs) and email it to the provided email account from the License Wizard.
You can either save the attachment to a local directory or copy the content between '===>' and '<===' in the email, then paste it into this page. Click "Next".
If all has been followed correctly, a message that the license has been imported successfully will appear.
Click finish and restart PEAKS.

2. Question: I’m following the registration procedure but I’m unable to complete it.

Answer. There may be a firewall causing you to not be able to connect to our license server. Not to worry, you can register without an internet connection. The first time PEAKS is run, the license wizard will appear automatically.

Select the second option, request license file (without internet connection). Click "Next".
Enter the necessary registration information. The email address entered here will eventually be used to send you a license file, different from a registration key. If you have purchased PEAKS and have a registration key, looks like PS*************, enter/paste this into the respective field, careful not to include any spaces. For those evaluating the software, request a license file by entering your instution's name in the corresponding lower field. Click "Next".
The window now offers the opportunity to "Save Request File" to our computer, by clicking on the button. Once saved, click "Next".
Transfer the license request file from this PC to another PC which does have an internet connection, perhaps via USB key or another removable storage device.
Using the PC with internet connection, logon to http://www.bioinfor.com/lcs20.
Select "I have the license request file. I want to register the software" and click "Next".
Click the "Browse" button to select the license.request file, type in the visual verification code and click "Next".
An automated BSI service will generate the license file (license.lcs) and email it to the provided email account entered from before.
After the license email is received on the PC with internet connection, save the attachment, license.lcs, as is and copy the file to the computer without an internet connection.
If you do not receive the license.lcs file in your inbox, please check your junk mail folder.
In the license wizard on the computer without an internet connection, click the 'Browse' button below to select the license.lcs file and click "Next".
If all has been followed correctly, a message that the license has been imported successfully will appear.
Click finish and restart PEAKS.

3. Question: What are the recommended/maxmimum performance configurations for PEAKS?

Answer. On this page, you will find the recommended configurations for PEAKS Studio and PEAKS Online, desktop/server solutions respectively, as well as ideal configurations.

4. Question:How do I optimize PEAKS Studio for Windows 7/Windows XP 64 bit?

Answer. Go to the link: http://java.sun.com/products/archive/j2se/6u18/index.html. You can also enter 'java jre update 6u18' into Google and search for it that way.

From there, select 'download JRE'
Beside platform select 'Windows x64'
Don't worry about registering, click continue
Click on jdk-6u18-windows-x64.exe
Select save
Run the downloaded installer. This should save the file to: C:\Program Files\Java\jre1.6.0_18
Open PEAKS then go to Preferences -> Performance
Click on the check boxes beside 'Start Client Separately' and 'Start Compute Node Separately' to turn them on.
Ensure that the Client node is set to run on the JRE that comes with PEAKS. The path should be: C:\PeaksStudio5.3\jre\bin
Keep the Client node memory maximum below 1200 MB
Click browse beside 'Compute Node JRE Binary folder'
Set the path to C:\Program Files\Java\jre1.6.0_18\bin
From there you will be able to allocate as much memory to the compute node as possible, up to the physical maximum of your RAM.
When you are finished the performance parameters should look something like the attached screenshot.

5. Question: How can I change my memory heap size for Java so that PEAKS has more memory to use?

Answer. The default of 1024 MB tells the Java Virtual Memory (which runs PEAKS) to run with 1024 MB of memory. To increase the JVM and determine the highest optimal value for your own computer. Click the "Memory Utility" icon, found in your installation directory. Often the Start Menu will host this feature, with the other PEAKS applications. Once the window is open, in the designated field, enter the preferred settings, then hit Apply. Some trial and error may be needed as Java will not start if you set the value to be too large for your system.

6. Question:Can PEAKS read my raw data?

Answer. PEAKS can, please check our data types section for how to load data from your instrument.

7. Question: Why do I get the error message, ‘Could not create the Java virtual machine’ when trying to start PEAKS?

Answer. That error message comes up if the maximum heap size for the interface java virtual machine is too high to start. It usually appears after someone has changed some performance settings. It can also appear if you have installed some software which causes the computer to have to multi-task heavily. Check the memory utility under start menu -> PEAKS Studio -> Memory Utility. Try a value around 1000 MB.

8. Question: How do I connect to the Mascot server through PEAKS for inChorus?

Answer. In Preferences, go to “Search Engine”, then “Mascot Settings”.

Enter this in the ‘Hostname (or IP address):’ text box.
Enter the ‘Port:’ number, the default is 80.
Enter the virtual directory, the default is ‘/mascot’.
Select your version number in the ‘Version’ drop down menu.
If you need to use a user name and password to login, you can enter them in the sections below the version number.
Check the "Save Password" box so you do not have to enter it every time.
Click the "Apply" button to save any changes you have made.
In the event that Mascot is installed on the local computer, type "localhost" in the Hostname (or IP address) field.

9. Question: How do I configure Sequest for PEAKS inChorus?

Answer. Just open up Preferences -> Search Engines -> Sequest.

Click the "Browse" button to tell PEAKS where to find the search engine.
You must also browse your computer to find the location of the "Default Sequest Parameter File (.params)" as well as the "Sequest Result Output Folder".
Click the "Apply" button to save any changes you have made.

10. Question: I am using a computer with regional settings not set to English. Will this cause complications?

Answer. We strongly recommend switching your computer’s locale to US/English when using PEAKS. There have been cases where the number formatting, specifically utilizing a comma rather than a decimal has affected the results. For example 1.000,99 may appear in a German locale computer instead of 1,000.99 in English locale. PEAKS will misinterpret this value as 100099.00.

11. Question: Can PEAKS run two fragment type, for example, CID and ETD??

Answer. The answer is yes. When you create a project from the RAW data, make sure select the right instrument type (e.g. choose FT-trap (ECD-CID) if your RAW data is from FT-Trap or Orbi-trap instrument and it also contains both CID and ETD fragment modes). PEAKS will analyze the spectrum according to its fragment mode. You can also run CID or ETD separately, just remember to correctly designate the instrument types during the project creation.

12. Question: My instrument is not listed in list of supported instruments. Will PEAKS accept my data?

Answer. Yes, provided the data is in one of the supported data formats, it certainly can be read by PEAKS. To add the new instrument to the list of supported instruments select Configuration > Instrument > New Instrument. For this example let's imagine you are looking to add a Thermo Velos to the list of instruments. Under the Basic Options:

Give the instrument a name: Velos
Manufacturer: Thermo Scientific
Ion Source: ESI (Nano-Spray)
MS Precursor Scan: FT-ICR/OrbiTrap
Fragmentation Type: High Energy CID (y and b ions)
MSn Product Scan: FT-ICR/Orbitrap

If you have a prefered set of Adavanced Options you wish to be entered as standard tolerances, enter them here. Click the Add/Update button and it will be added to your list of instruments for future use.

De Novo Sequencing

1. Question: What do the TLC and ALC scores in my de novo results mean?

Answer.

Local Confidence is the confidence that a particular amino acid is present in the de novo peptide at a particular position. It is presented as a percentage.
Total Local Confidence (TLC) is the sum of the local confidence scores (0 to 1) from each amino acid in the peptide sequence.
Average Local Confidence (ALC) is the average of the TLC. It is TLC divided by the number of amino acids in the peptide sequence.

2. Question: What is the cut off for a good de novo result?

Answer. Usually you will find good peptides at 55% and above. This does not mean that the whole sequence is the correct sequence when it comes to de novo. This is because the beginning and end of the spectrum are much more difficult to interpret when it comes to de novo sequencing due to the high mass/low mass of the ions produced by these fragments. This is why we added the colour coding to the results so you can see where in the sequence there is higher confidence.

3. Question: How to remove low scoring amino acids from the ends of de novo sequence tags before exporting?

Answer. You can definitely chop off the low scoring amino acids from the ends of the de novo sequence tags. To do this, click on the score threshold slider button (green, yellow, red symbol at the top of the de novo results). Use the slider to set a score threshold and any amino acid below that threshold will be reduced to its mass in Daltons. Once you export the tags, the last column in the excel export will follow this confidence threshold. De Novo Tags

4. Question: What are the differences between PEAKS and other de novo sequencing programs?

Answer. Bottom line: result quality. PEAKS consistently gives more accurate peptide sequences and with better confidence than other programs. This is a result of PEAKS' global optimization algorithms and sophisticated scoring schema.

Database Search (PEAKS DB)

1. Question: What is the meaning of the -10lgP Score?

Answer. The -10lgP score is simply a p-value on an increasing scale. For example, a p-value of 1% equals a -10lgP of 20. For a given score x, its equivalent P-value is the probability that a false identification has a score greater than or equal to x. So, generally speaking a -10lgP of 20 has a 1% FDR. For more information on the scoring system see the following technical note.

2. Question: How should I format the header to use a custom database?

Answer. PEAKS only requires the database to be in FASTA format, and the header should contain only standard ASCII characters.

3. Question: PEAKS DB: No results, what happend?

Answer. Likely the database was simply not located by PEAKS. In PEAKS, go to Configuration then Database. Make sure you have the correct FASTA database type selected in the drop down menu and the correct database path selected in the path text box.

4. Question: Why am I geting taxa A when I set taxa B?

Answer. PEAKS requires the taxonomy files downloaded from the database source to be in their compressed form. Most often, users forget to unzip the taxonomy files.
If the taxa files are in order, the files may be corrupt, as indicated by a similar error message:

22-feb-2011 16:25:24 com.bsi.peaks.io.i a

GRAVE:

java.util.zip.ZipException: error in opening zip file

at java.util.zip.ZipFile.open(Native Method)

(Unknown Source)

at com.bsi.peaks.io.databaseio.o.a(Unknown Source)

(Unknown Source)

at com.bsi.peaks.io.databaseio.m.d(Unknown Source)

at com.bsi.peaks.io.databaseio.l.a(Unknown Source)

at com.bsi.peaks.system.server.c.b(Unknown Source)

at com.bsi.peaks.system.server.c.a(Unknown Source)

at com.bsi.peaks.system.rmi.b.a(Unknown Source)

at com.bsi.peaks.system.rmi.e.a(Unknown Source)

at com.bsi.peaks.system.server.z.a(Unknown Source)

at com.bsi.peaks.system.server.cb.run(Unknown Source)

at java.util.concurrent.ThreadPoolExecutor$Worker.runTask(Unknown Source)

at java.util.concurrent.ThreadPoolExecutor$Worker.run(Unknown Source)

at java.lang.Thread.run(Unknown Source).

5. Question: How does PEAKS handle PTMS with a neutral loss mass?

Answer. For PTMs with a neutral loss mass, PEAKS will search for both the monoisotopic mass of the modified amino acid as well as the neutral loss mass. PEAKS does not require the neutral loss mass to be there in order for the modified amino acid to be identified. It does help if it is present though.

inChorus (Multi-Engine Database Search)

1. Question: What do the inChorus percent scores mean?

Answer. The peptide percent score reflects the quality of the peptide-spectrum match taking into account all search engines used. The score is calculated in accordance with the empirical calculation used in Peptide-Prophet.
The protein percent score is calculated from the peptide percent scores. The peptide percent scores are combined by a weighted sum for each protein. Then, the PeptideProphet method is then applied to the whole set of proteins to get the final protein probability scores.

2. Question: Why is the ppm mass error so high for my OMSSA result?

Answer. OMSSA will try all the charge states you set when setting up the search. If it finds a good match with a different charge than what is read in from the mass spec the mass error will be very high because the theoretical mass will be from the charge state found with OMSSA and the actual mass will be from the mass spectrometer. See image for more details. OMSSA

SPIDER (Sequence Tag Homology Search)

1. Question: In SPIDER, what is the different between Tag Match and Homology Match?

Answer. Tag match will search the database with your de novo peptides and take into account common de novo errors. Tag match allows you to take into account fixed modifications. Homology match will search the database with your de novo peptides taking into account all possible mutations and the de novo local confidence values. It will also take information from both the database sequence and de novo sequence to reconstruct the best possible match. Homology match allows you to take into account fixed and variable modifications.

2. Question: What constitutes a good SPIDER match?

Answer. A confident SPIDER assignment requires a few things:

high SPIDER score (30 or greater)
low RSD (0.2 or lower, can be higher in some cases)
good quality de novo result (check spectrum alignment)
homologous protein must be from a homologous species

Here is some more information on the SPIDER score. The SPIDER score is actually a measure of the similarity between the de novo tag and the database hit weighted by the quality of the de novo result. The higher the score the better (this is not a percentage). Below you will notice a graph from some research that was done by one of our developers on a known protein sample from an LCQ experiment. It shows that a SPIDER score of 30 matches the correct sequence 75% of the time, 35 matches the correct sequence 85% of the time, and 40 or greater matches the correct sequence 99% of the time.
SPIDER, sequence tag homology searching

RSD (relative standard deviation) is a direct measure of the similarity between the de novo result and the database entry. The assignment is split into four sections and is calculated for each section. The value shown in the result table is an average of the four. Further, here is an example publication where SPIDER was used well:
SPIDER Stressad Aptation Table 2 Source: Farinha AP, Irar S, de Oliveira E, Oliveira M, Pagès M. Novel clues on abiotic stress tolerance emerge from embryo proteome analyses of rice varieties with contrasting stress adaptation. Proteomics. 2011 March 31.

Quantification

1. Question: What does "-1" mean in my iTRAQ/SILAC results?

Answer. "-1" means that a ratio was found but it was not derived from a high scoring peptide. If you want the ratio to be counted you can change the minimum and maximum scores of a peptide that are included in the ratio calculation.

2. Question: Why can't I perform label free quantification?

Answer. The most likely reason is users to have at least two samples, the same number of fractions in each sample. Also, label free quantification works best if label free quantification is performed on the combined protein ID results of all the samples.

3. Question: How can I use 3D View?

Answer. As this may be considered an advanced monitor feature, the 3D View may only work for computers that have newer video cards. If it is turned on for an older computer, it may cause a JVM crash, resulting in PEAKS shutting down. For computers with the necessary hardware, the feature to turn on and off the 3D View can be found in Preferences -> Performance. When the tick box has a check mark inside, the 3D View is enabled; when it is clear, the 3D View is disabled.

"Thanks a lot, I more and more like the power of PEAKS, it is simply great." Manfred Raida, Biomedical Sciences Institutes

Installation/Configuration

De Novo Sequencing

Database Search (PEAKS DB)

inChorus (Multi-Engine Database Search)

SPIDER (Sequence Tag Homology Search)

Quantification

Installation/Configuration

De Novo Sequencing

Database Search (PEAKS DB)

inChorus (Multi-Engine Database Search)

SPIDER (Sequence Tag Homology Search)

Quantification

"Thanks a lot, I more and more like the power of PEAKS, it is simply great."

Manfred Raida,
Biomedical Sciences Institutes