Build library question - new user

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Build library question - new user William  2012-08-20 16:40
 
Hi Brendan,
I am new to proteomics though not new to MS. I wish to use Skyline to do some targeted proteomics work with our Agilent QQQ. I have followed the tutorial entitled "Skyline Targeted Method Editing" to familiar with the process. However, I am still not clear how to build my own library. For example, currently I only work with one protein (P16435) and sequence / peptide info can be easily got from PeptideAtlas (https://db.systemsbiology.net/sbeams/cgi/PeptideAtlas/GetProtein?atlas_build_id=348&protein_name=P16435&action=QUERY). But what to do next to build the library? Sorry for the newbie's amateur question, it prevent me from going further with Skyline.
 
 
Brendan MacLean responded:  2012-08-20 17:25
Hi Y. Gu,
It is not immediately clear to me that you need to build a library of your own. You may want to start by downloading a library from PeptideAtlas, NIST or the GPM. There are links for all of these in the Edit Library form, which you may have seen in the tutorial you described. You can find the same links at the bottom of the Skyline home page:

https://skyline.gs.washington.edu/labkey/project/home/software/Skyline/begin.view

If you do not find enough peptides or high enough quality spectra for the peptides or modification states you seek in these libraries, then you would want to build your own library. You would need to start with some runs on a full-scan instrument like an ion trap or a Q-TOF, which you then search using a peptide search engine (e.g. X! Tandem, OMSSA, Mascot, etc.) Once you have searched MS/MS spectra that you feel confident represent the peptides and modification states you are interested in, then you can start thinking about building a library for Skyline to use, and I will be happy to point you in the right direction then. But, it is not sounding like you are all the way to this stage. And, it may not be necessary, if the public libraries contain what you need.

Good luck with your first steps in proteomics. Thanks for choosing Skyline for your investigation.

--Brendan
 
William responded:  2012-08-20 18:09
Thanks for your prompt response. I have downloaded Human Q-TOF splib from PeptideAtlas as well as other 2 formats (msp and nist), but after unzip, it doesn't look like the same file format as the example in the tutorial (where there are 3 sub-folders entitled "FASTA", "Library" and "Yeast_atlas" with corresponding files underneath). Instead, for example, there are 4 files with extension of .pepidx, .spidx, .splib and .sptxt in splib format.
That's why I am lost. Could you pls let me know how should I do with those files to match the procedure in the tutorial??
Cheers, William
 
Brendan MacLean responded:  2012-08-20 18:28
Hi William,
You should be able to use the .sptxt or .msp files in the Edit Library form, as well as .hlf from the GPM or .blib from a library build.

If you click the Browse button in the Edit Library form, you will see these extensions listed. See the attached PowerPoint slides.

Hope that helps. Good luck creating your first targeted proteomics method.

--Brendan
 
William responded:  2012-08-20 20:28
Thanks, I've added the public library as instructed (on p9 of the tutorial too I think). Then how can I pick up the protein I need (for example: P16435) from the library and get the peptide/transition prediction? The tutorial doesn't seem to cover from there.
-William
 
Brendan MacLean responded:  2012-08-20 20:50
See

- Pasting FASTA Sequences (p. 5)
- Inserting a Protein List (p. 11)
- Creating a Background Proteome File (p. 3) and Protein Name Auto‐Completion (p. 16) and Protein Description Auto‐Completion (p. 17).

Are you sure we are talking about the same tutorial (Skyline Targeted Method Editing)? Maybe it is time for a review. There is a lot of information in there. Read it through thoroughly, and you will gain a much better understanding of how to use Skyline for your desired task.

--Brendan
 
William responded:  2012-08-20 21:05
Thanks, that's my question here. In tutorial, the FASTA.txt and protein list file were given, do you me I have to create these files manually?
 
Brendan MacLean responded:  2012-08-20 21:22
There are on-line sources from which you can download them. In the case of your single protein, you could simply copy the sequence from the PeptideAtlas link you provide above, and then add a title line starting with >. Like:

>P16435 NCPR_HUMAN NADPH--cytochrome P450 reductase OS=Homo sapiens GN=POR PE=1 SV=2
MGDSHVDTSS TVSEAVAEEV SLFSMTDMIL FSLIVGLLTY WFLFRKKKEE VPEFTKIQTL TSSVRESSFV
EKMKKTGRNI IVFYGSQTGT AEEFANRLSK DAHRYGMRGM SADPEEYDLA DLSSLPEIDN ALVVFCMATY
GEGDPTDNAQ DFYDWLQETD VDLSGVKFAV FGLGNKTYEH FNAMGKYVDK RLEQLGAQRI FELGLGDDDG
NLEEDFITWR EQFWPAVCEH FGVEATGEES SIRQYELVVH TDIDAAKVYM GEMGRLKSYE NQKPPFDAKN
PFLAAVTTNR KLNQGTERHL MHLELDISDS KIRYESGDHV AVYPANDSAL VNQLGKILGA DLDVVMSLNN
LDEESNKKHP FPCPTSYRTA LTYYLDITNP PRTNVLYELA QYASEPSEQE LLRKMASSSG EGKELYLSWV
VEARRHILAI LQDCPSLRPP IDHLCELLPR LQARYYSIAS SSKVHPNSVH ICAVVVEYET KAGRINKGVA
TNWLRAKEPA GENGGRALVP MFVRKSQFRL PFKATTPVIM VGPGTGVAPF IGFIQERAWL RQQGKEVGET
LLYYGCRRSD EDYLYREELA QFHRDGALTQ LNVAFSREQS HKVYVQHLLK QDREHLWKLI EGGAHIYVCG
DARNMARDVQ NTFYDIVAEL GAMEHAQAVD YIKKLMTKGR YSLDVWS

So, I have done this first one for you. Now all you have to do is copy and paste it into Skyline. Hopefully, you can see that this was pretty simple. You can also download FASTA files with the entire proteome for various organisms (this looks like a useful page http://www.matrixscience.com/help/seq_db_setup_IPI.html), and use these to create a background proteome file, as described in the tutorial.

--Brendan
 
adrien mombrun responded:  2016-06-07 06:55
Dear all,

I have the same problem as William

I downloaded NIST, SPLIB, MSP formats from peptide atlas (human) and skyline always tells me "not a valid library input file". I can't find any extension listed in the "add inpunt file" window on the web !!

Thx for any help.

Adrien
 
Kaipo Tamura responded:  2016-06-07 12:13
Hi Adrien,

You should be able to add .msp files as libraries to Skyline through Settings > Peptide Settings... > Library > Edit list... > Add..., giving the library a name and browsing for the path of the .msp file. If that is not working for you, would you mind sharing the link to the msp file that you downloaded?

Thanks,
Kaipo
 
adrien mombrun responded:  2016-06-08 01:12
Ok I was using the "build" buton and not the "edit list > add" buton. Now it works. What is the difference between the two approaches ??
 
Kaipo Tamura responded:  2016-06-08 02:16
Hi Adrien,

The build button is used for when you want to build a spectral library from the result files of a search pipeline. You can see the supported formats here: https://skyline.gs.washington.edu/labkey/wiki/home/software/BiblioSpec/page.view?name=BlibBuild
Formats like .sptxt and .msp can be used directly as spectral libraries. Hope that makes sense.

Thanks,
Kaipo