Building a spectral library using msp

Building a spectral library using msp laura corveleyn  2021-02-02


I am trying to build a spectral library in Skyline using an msp file based on predicted spectra. Since we work with histones, we derivatize our peptides using propionylation, so all unmodified lysines and N-terms are blocked with a propionyl group. Therefore, Propionyl (K) and Propionyl (N-term) are fixed modifications, unless another variable modification is present. However, Skyline does not seem to recognize the modification "Propionyl" from the msp, although I also defined it as a modification in the peptide settings. All other modifications (e.g. Acetyl, Dimethyl,..) that I defined, are used correctly in the library. After some research, we figured out that Skyline recalculates the precursor m/z for each peptide (based on its sequence) and doesn't use the one that is stated in the msp. So for a peptide that carries a propionyl group, the m/z is recalculated as if the peptide would be unmodified because Skyline does not recognize the propionyl.

In the attachment you can find a short example of a predicted msp and the Skyline document with the corresponding library I built with this msp. In the msp you can see that the Propionyl group in the header is stated in the exact same way other modifications are, so I don't understand where the problem is.

Thank you in advance for helping me out!



Nick Shulman responded:  2021-02-02
There is a limited set of modifications that Skyline understands when reading a .msp file. You can find that list of modifications here:

Where did you get this .msp file?

Could you use a .blib file instead? With .blib files, Skyline does a lot of work to try to figure out what modifications in your document have the same mass as the modifications in your library, but for .msp files, you are limited to the set of modifications in that list.

I am not the expert on .msp files, so I am not sure why Skyline has such a limited list, or what it would take to add things to that list.
-- Nick
laura corveleyn responded:  2021-02-02
Hi Nick,

Thanks for the quick response! This explains a lot. Reading Victor's request, I see we have the same problem. Our .msp file is also created in-house (using predictions) and we can't use a .blib file instead. It would indeed be very helpful if someone else of your team could help us out with this one.

Kind regards,

Brian Pratt responded:  2021-02-02
It seems like we should be able to do better: "although I also defined it as a modification in the peptide settings" is a perfectly logical thing to expect to work since the MSP reader is built into Skyline, unlike the various blib converters.

I'll open a ticket on this.


Brian Pratt
laura corveleyn responded:  2021-04-08

I was wondering if there are any updates on this issue?

Many thanks!

Kind regards,

Brian Pratt responded:  2021-04-08
None yet, but I've added you to the notifications list for the issue at so you'll know when we get to it. But it's never a bad idea to ask - squeaky wheels and grease, you know.

laura corveleyn responded:  2021-07-26
Hi Brian,

I see that I don't have access anymore to the issue update. Does this mean this has been resolved or are there any other updates on this? ;)


Kind regards,

Brendan MacLean responded:  2021-07-26
That was the wrong URL to put on the support board. You want to check this one:

I don't see that anything has changed. You should have gotten email if it had.