SAV recognition by speclib builder

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SAV recognition by speclib builder dkueltz  2020-06-27
 

Hi Brendan et al.
I am using SPIDER within PEAKSsuiteX to identify allelic variants that manifest themselves at the protein level as single amino acid substitutions (SAVs). These can be exported from PEAKS to pep.xml + mzxml files that Skyline uses to built spectral libraries from DDA data. However, unlike unmodified peptides and regular PTM peptides, the SAV peptides are not recognized by the spec library builder. I was not able to open the mzxml files but the structure of SAVs is a bit different from that of regular PTMs in the pep.xml files (see below). I would be happy to send you a pair of pep.xml + mzxml files that can be used for speclib construction with Skyline and contain umnmod. PTM, and SAV peptides. Is there any way that I could import these SAV peptides into Skyline spectral libraries such that I can quantify the stoichiometry of allele-specific expression (ASE) in different biological contexts? If this is not possible yet, do you have any plans to implement this capacity into Skyline in the future?
Thanks,
Dietmar
Below is part of a pep.xml file that shows 3 entries, one each for a regular PTM, SAV, and unmod. peptide... in this particular example the PTM and SAV are actually the same - deamidation of Q to E - but there are many other instances with unique SAV mass changes that do not match a conventional PTM and can be interpreted unambiguously...
<spectrum_query spectrum="DK0020-B1B_2-A,1_01_2365.d" start_scan="2626" end_scan="2626" precursor_neutral_mass="1515.7789" assumed_charge="3" index="1303">
<search_result>
<search_hit hit_rank="1" peptide="LSNGTTKPQTNGVAK" calc_neutral_pep_mass="1515.7893" massdiff="4.7" num_tot_proteins="1" protein="XP_033982937.1" protein_descr="mucin-5AC [Trematomus bernacchii]" protein_mw="124905.71">
<modification_info modified_peptide="LSNGTTKPQ(+.98)TNGVAK">
<mod_aminoacid_mass position="9" mass="129.04259"/>
</modification_info>
<search_score name="-10lgP" value="44.82"/>
<search_score name="confidence" value="0.99"/>
<search_score name="PeaksScore" value="99.8"/>
<analysis_result analysis="reconstruction">
<tags denovo_tag="LSNGTTKPQTNGVAK" reconstructed_tag="LSNGTTKPQTNGVAK"/>
</analysis_result>
</search_hit>
</search_result>
</spectrum_query>
<spectrum_query spectrum="DK0020-B1B_2-A,1_01_2365.d" start_scan="2935" end_scan="2935" precursor_neutral_mass="1515.7789" assumed_charge="3" index="1304">
<search_result>
<search_hit hit_rank="1" peptide="LSNGTTKPQTNGVAK" calc_neutral_pep_mass="1515.7893" massdiff="4.7" num_tot_proteins="1" protein="XP_033982937.1" protein_descr="mucin-5AC [Trematomus bernacchii]" protein_mw="124905.71">
<modification_info modified_peptide="LSNGTTKPE(sub Q)TNGVAK">
<aminoacid_substitution position="9" orig_aa="Q"/>
</modification_info>
<search_score name="-10lgP" value="41.80"/>
<search_score name="confidence" value="0.95"/>
<search_score name="PeaksScore" value="95.8"/>
<analysis_result analysis="reconstruction">
<tags denovo_tag="LSNGTTKPETNGVAK" reconstructed_tag="LSNGTTKPETNGVAK"/>
</analysis_result>
</search_hit>
</search_result>
</spectrum_query>
<spectrum_query spectrum="DK0020-B1B_2-A,1_01_2365.d" start_scan="2596" end_scan="2596" precursor_neutral_mass="1088.5857" assumed_charge="3" index="1309">
<search_result>
<search_hit hit_rank="1" peptide="YTRPTPVQK" calc_neutral_pep_mass="1088.5978" massdiff="0.4" num_tot_proteins="1" protein="XP_034007142.1" protein_descr="ATP-dependent RNA helicase DDX3X-like [Trematomus bernacchii]" protein_mw="80330.54">
<search_score name="-10lgP" value="41.08"/>
<search_score name="confidence" value="0.97"/>
<search_score name="PeaksScore" value="96.3"/>
<analysis_result analysis="reconstruction">
<tags denovo_tag="YTRPTPVQK" reconstructed_tag="YTRPTPVQK"/>
</analysis_result>
</search_hit>
</search_result>
</spectrum_query>

 
 
dkueltz responded:  2020-06-30

Hi again,
I figured out a workaround for this issue - so please disregard the request. Workaround: Define all SAVs as regular PTMs in PEAKS, which then displays them in the conventional PTM format in mzxml and pep.xml files. That way I can add any SAVs I am interested in to the Skyline library builder/explorer.
Thanks!
Dietmar