Intact proteins+MRM z1854947  2020-06-12
 

Hello,

I am new to the skyline family.

My current project is specifically focusing on Top down proteomics. I am separating and quantifying intact proteins using Shimadzu Triple-Quadrupole 8045 by optimizing MRM methods.

Can anyone help me by listing the specific tools/setting which will be helpful for my kind of work? Most of the tools on Skyline that I learnt are for tryptic digested peptide.

Also, on the instrument, my files are generated with .mzxlm extension. How can I upload these files?

Thank you.

 
 
Brendan MacLean responded:  2020-06-12

Skyline can import both Shimadzu native .lcd files and .mzXML files. So, you should be set for importing your data.

Skyline supports specifying any amino acid sequence as a "peptide". It need not be a true peptide that underwent protease cleavage. This has been used to study larger neuropeptides which also experience no protease cleavage, and you can just add an arbitrary list of synthetic peptides with no relation to a protein and no implied cleavage. You can either just paste these into Skyline as a line-sperated list or you can use Edit > Insert > Peptides and past into the grid you are presented. The fact that Skyline will consider your list of targeted sequences a "peptide list" is unimportant. It is really a list of targeted amino acid sequence molecules.

When you paste "peptides" like this, you can also specify a charge state by adding pluses to the end of the sequence, and after charge 4, you can use the format Skyline uses:

<long AA sequence>++++++++++++
<long AA sequence>, +12

You may also need to adjust your Transition Settings - Instrument tab Max m/z value, if these intact proteins have high m/z values, though, not if they have high enough charge states to fall within the normal range.

Hope this helps. Please keep us posted on any issues you find performing your experiment.

--Brendan

 
Nick Shulman responded:  2020-06-12
There is no way to choose "no enzyme" in Skyline, so, for top-down proteomics in Skyline, the trick is to avoid the menu item such as "Import FASTA" where Skyline will try to digest the protein sequences.

Instead, the way that you can get your protein sequences into Skyline is by using the menu item:
Edit > Insert > Peptides
and put the protein sequence in the "Peptide Sequence" column.

Also, you will need to go to:
Settings > Transition Settings > Filter
and add the precursor and product charge states that you are interested in.

I have never heard of mzxlm files. The Shimadzu files that we know how to deal with have the extension ".lcd".

Skyline also knows how to read .mzML and .mzXML files.

After you have added your peptide sequences to your document, the menu item to tell Skyline to read chromatograms from a raw file is "Import > Results".
-- Nick