If you send us your Skyline document and one or more of your raw files we will be able to tell you what is going wrong.

In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.
The "Share Document" dialog also gives you the option to include raw files in the zip file that is being created, or your can send them to us separately.

Files which are less than 50MB can be attached to this support request. You can always upload larger files here:
https://skyline.ms/files.url

The usual reason that it says "Chromatogram information unavailable" with PRM data is that there really were no MS2 spectra whose isolation m/z matched the m/z of the precursor in your document. There is a setting "Method match tolerance m/z" at "Settings > Transition Settings > Instrument" which controls how closely those numbers have to match up. You can sometimes get chromatograms to show up by increasing setting that to a larger number and then telling Skyline to extract chromatograms again using the "Reimport" button at "Edit > Manage Results".

Another thing that could cause the chromatogram to be unavailable would be if you had set the "Explicit Retention Time" on the molecule to a value which is outside of the range over which there were matching MS2 spectra. Skyline always discards extracted chromatograms whose time range does not overlap with the explicit retention time.

After we see the files that you send us we will probably be able to tell you exactly what is going wrong.
-- Nick

Hello Nick,

Thanks for the response. I am attaching my PRM raw and Skyline zip file for your kind perusal.
I really appreciate your assistance.

Thanks
VC

It sounds like you intended to attach some files, but nothing actually go attached.
This often happens if the files that you were attaching were larger than the 50MB limit.
You can upload larger files here:
https://skyline.ms/files.url
-- Nick

Thank you; I submitted my files using the URL you provided.
File name: Raw data_VC


Thanks
VC

Thank you for sending that .zip file.
I can see the two .raw files in there, but I am not sure what the other thing in there is. It's not a Skyline document.

Can you use the "File > Share" menu item in Skyline to create a .zip file and upload that .zip file?
-- Nick

Hello
Are you looking for this Skyline document as attached.

No, that's not the right file.

In Skyline you must use the menu item:
File > Share
to create a .zip file and then send me that .zip file.

-- Nick

Thanks for the clarification and please find the attached zip file.


Thanks
VC

Unfortunately, the Skyline document that you sent me is empty.

What I meant was, when you are using Skyline and you are see the message that says "chromatogram information available" you should use the menu item:
File > Share
to create a .zip file.

-- Nick

Sorry for that. Please find the attached file.

Thanks
VC

Thank you for sending that Skyline document.

The precursor m/z's of the Targets in your Skyline document are different from the m/z's that were isolated in your .raw files.

These were the m/z's that were isolated in your .raw files:
906.12622, 923.11719, 986.41461, 1011.7439, 1014.4005, 1022.9308, 1022.9322, 1033.6768,
1033.9196, 1070.1836, 1106.6979, 1108.7747, 1114.214, 1133.4545, 1150.9791, 1161.4667,
1186.988, 1201.2769, 1207.8325, 1223.504, 1230.4882, 1232.5134, 1234.2417, 1236.9073,
1270.5046, 1314.8811, 1327.5197, 1329.5433, 1352.2003, 1363.9045, 1400.8837, 1402.5696,
1426.5767, 1475.5952, 1485.2822, 1548.2853, 1582.6481, 1601.3698, 1645.6534, 1693.6711

However, the precursor m/z's of the things in your Skyline document are different (see attached screenshot "TargetsTree.png")

Do you have any information about the instrument method that was used to collect the data in your .raw files, and what peptides the method was targeting?
-- Nick

I really appreciate your assistance. How to fix this issue.

My Instrument details and methods are as followed,
Thermo Fusion Orbitrap
HCD_MS2_PRM
Targets: Glycopeptides


Thanks
VC

Can you tell me more about the glycopeptides that you were targeting? Do you have a list of those glycopeptides?

The peptides in your Skyline document are all ordinary unmodified peptide sequences. You need to tell Skyline about the modifications that are on those peptides so that they will have the correct m/z's.

Here is a webinar which talks in general about working with modifications in Skyline:
http://skyline.ms/webinar10.url
-- Nick

Thanks please refer the list of glycopeptides that we are looking for.
MVSHHNLTTGATLINE
NLFLNHSE
KVVLHPNYSQVD
KVVLHPNYSQVDIGLIK


Thanks
VC

A glycopeptide has a sequence of amino acids, but it also has post-translational modifications (PTMs) attached to one or more of the amino acids.
Do you know which PTMs are attached to your peptides and which particular amino acids they are attached to?
-- Nick

Yes, PTMs are attached in amino acid "N" in all the peptides in the listed below.
MVSHHNLTTGATLINE
NLFLNHSE
KVVLHPNYSQVD
KVVLHPNYSQVDIGLIK

Thanks
VC

Do you know the name and/or the chemical formula of the PTM that is applied to those asparagine residues?
In Skyline you should go to "Settings > Peptide Settings > Modifications" and then push the "Edit List..." button next to "Structural Modifications" and then tell Skyline about the modifications.
Then you will need to right-click on your peptides in the Targets tree and tell Skyline which residues are modified.

I think this topic is fairly well covered in the Modifications Webinar:
http://skyline.ms/webinar10.url
-- Nick

I appreciate the explanations. I've watched the modifications Webinar and added all of my modifications by following your directions.
Unfortunately, none of the peptides below include the precursor number I was hoping to find.
MVSHHN[+2351.851]LTTGATLINE
NLFLN[+3007.058]HSE
VVLHPN[+2350.830]YSQVD
VVLHPN[+3008.078]YSQVDIGLIK


For your good examination, kindly find the Skyline documents attached.


Thanks
VC

Things will work a little better in Skyline if you are able to tell Skyline the chemical formula of those modifications.
However, you can simply tell Skyline the mass of the modification as I have done in the attached screenshot "MassOnlyModification.png".

Your modification with the mass of 2350.830 might be the one called "dHex(1)Hex(5)HexNAc(4)NeuAc(2) (N)" with the chemical formula H146C90N6O65.
Skyline version 23.1 does not know about that modification, but our beta release called "Skyline-daily" has an updated list of Unimod modifications and does know about it.

I made a document with these peptides and extracted chromatograms from the data that you sent me.
You can open the attached file "NickSkylineDocument.sky.zip" in Skyline.
The peptides "MVSHHN[+2351.851]LTTGATLINE" and "VVLHPN[+3008.078]YSQVDIGLIK" look pretty good.
The other peptides do not look good at all which might mean that they are not present in your sample, or it might mean that there is something else wrong.
-- Nick

Its really helpful. I appreciated for your great assistance.

Hello Nick,
How can I obtain the glycan structure's chemical formula? Is there a conversational tool available?
Thanks in advance.


VC

You might be able to use the NIST Glyco Mass Calculator:
https://www.nist.gov/mml/biomolecular-measurement/mass-spectrometry-data-center/nist-glyco-mass-calculator
-- Nick

I built all of the targets and chemical compositions, thanks for the link. I am unable to obtain chromatogram information, though.
For your reference, kindly locate the Skyline document and my target list.
Please give me your valuable suggestions.

Thanks
VC

One thing that I think it messing you up is that at "Settings > Peptide Settings > Modifications", you have listed your glycan modifications in the lower listbox where it says "Isotope modifications".
Your glycan modifications definitely belong in the upper list box where it says "Structural modifications".

I have moved the modifications into the correct place in the following attached Skyline document "test1.sky.zip".

You have added a lot of modifications at "Settings > Peptide Settings > Modifications" and all those extra modifications might be confusing you.
I would recommend that you go to:
Tools > Options > Miscellaneous
and push the button "Clear all saved settings".

After you do that you can open the attached Skyline document.
-- Nick

Thanks for the clarification. I utilized the updated file you sent with me and I followed your comments.
I am unable to locate every changed site indicated in the target peptide list provided below, though. Rather, I've only identified two peptides, which yield two precursors (VVLHPN[+2350.8]YSQVD & VVLHPN[+3008.1]YSQVDIGLIK). Why this target list showing only two precursor? Is this usual or I need to change anything?.

Missing targets precursor
MVSHHN[+2204.77244]LTTGATLINE
MVSHHN[+2204.77244]LTTGATLINE
MVSHHN[+2351.85075]LTTGATLINE
MVSHHN[+2351.85075]LTTGATLINE
MVSHHN[+2350.83035]LTTGATLINE
MVSHHN[+2716.98295]LTTGATLINE
MVSHHN[+2716.98295]LTTGATLINE
MVSHHN[+3008.07836]LTTGATLINE
MVSHHN[+3008.07836]LTTGATLINE
MVSHHN[+2863.04086]LTTGATLINE
MVSHHN[+3154.13627]LTTGATLINE
NLFLN[+2059.73493]HSE
NLFLN[+2350.83035]HSE
NLFLN[+2424.86713]HSE
NLFLN[+2715.96255]HSE
NLFLN[+2715.96255]HSE
NLFLN[+3007.05796]HSE
NLFLN[+3082.11514]HSE
NLFLN[+3082.11514]HSE
NLFLN[+3227.15265]HSE
VVLHPN[+2350.83035]YSQVD
VVLHPN[+2350.83035]YSQVD
VVLHPN[+2424.86713]YSQVD
VVLHPN[+2715.96255]YSQVD
VVLHPN[+3008.07836]YSQVD
VVLHPN[+3008.07836]YSQVD
VVLHPN[+2862.02046]YSQVD
VVLHPN[+2862.02046]YSQVD
VVLHPN[+3154.13627]YSQVD
VVLHPN[+3154.13627]YSQVD
VVLHPN[+3373.21056]YSQVD
VVLHPN[+3372.19016]YSQVD
VVLHPN[+3664.30598]YSQVD
VVLHPN[+3664.30598]YSQVD
VVLHPN[+3809.34348]YSQVD
VVLHPN[+3809.34348]YSQVD
VVLHPN[+3008.07836]YSQVDIGLIK
VVLHPN[+3008.07836]YSQVDIGLIK
VVLHPN[+1913.67702]YSQVDIGLIK
VVLHPN[+2935.03683]YSQVD


Thanks
VC

The "Precursor Charges" setting that you have at "Settings > Transition Settings > Filter" is "2, 3", so, by default, Skyline will only add the +2 and +3 precursors to your document.
Furthermore, Skyline will also only give you precursors whose m/z is between the "Min m/z" and "Max m/z" setting at "Settings > Transition Settings > Instrument" (50 - 1800 m/z).

If you would like to have different precursors in your document you could change the "Precursor Charges" setting at "Settings > Transition Settings > Filter".
Alternatively, you could right-click on a peptide and choose "Add Children".
In the child picker window that appears, you might need to first click the filter button in order to be able to select the missing precursor charge states.
-- Nick

Hello Nick,
I appreciate your suggestions.
I got work with precursors setting. However I want to include every glycan in the following list in my targets. Only two glycans (VVLHPN[+2350.8]YSQVD & VVLHPN[+3008.1]YSQVDIGLIK) were mentioned after my attempt. The objective is to quantify each glycan's unique structure.

Note: We attempted to add the list you suggested (Pick Children), and we were successful in doing so; nevertheless, I am unable to obtain the chromatogram for those lists. Please find the attached Skyline document for your kind perusal.

Glycan list
VVLHPN[+2350.83035]YSQVD
VVLHPN[+2350.83035]YSQVD
VVLHPN[+2424.86713]YSQVD
VVLHPN[+2715.96255]YSQVD
VVLHPN[+3008.07836]YSQVD
VVLHPN[+3008.07836]YSQVD
VVLHPN[+2862.02046]YSQVD
VVLHPN[+2862.02046]YSQVD
VVLHPN[+3154.13627]YSQVD
VVLHPN[+3154.13627]YSQVD
VVLHPN[+3373.21056]YSQVD
VVLHPN[+3372.19016]YSQVD
VVLHPN[+3664.30598]YSQVD
VVLHPN[+3664.30598]YSQVD
VVLHPN[+3809.34348]YSQVD
VVLHPN[+3809.34348]YSQVD
VVLHPN[+3008.07836]YSQVDIGLIK
VVLHPN[+3008.07836]YSQVDIGLIK
VVLHPN[+1913.67702]YSQVDIGLIK
VVLHPN[+2935.03683]YSQVD


Thanks
VC

I am not sure that I understand your question now.
Some of the modified peptides in your Skyline document are displaying the "Chromatogram information unavailable" message because there were no MS2 spectra in your .raw file which matched the precursor m/z.
In order to see chromatograms for those peptides you will need to create a new method that includes the peptide's precursor m/z and then collect new data on your mass spectrometer.
The Skyline menu item "File > Export > Isolation List" may be very helpful for creating methods for your mass spectrometer to collect data for your targets.

You might find some helpful information in the PRM tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_prm
-- Nick