Hello,
I am attempting a SILAC-digly search by adapting the built-in SILAC3-phospho workflow. My experiment has 2 swap replicates (ctr Heavy/mut Light and mut Heavy/ctr Light), so ratios should reverse between replicates. Instead, both replicates produce ratios in the same direction in combined_modified_peptide_label_quant.tsv (Fig. 1).
Workflow configuration:
- MSFragger: SILAC3-phospho defaults + variable mods K114.04293, K8.014199, R10.008269; missed cleavages = 3; mass tolerance = 10ppm.
- PTMProphet: K:114.04293 added in the Cmd line opts
- Quant (MS1): Light = K0;R0 / Heavy = K8.014199;R10.008269
I have noticed 2 things in the combined_modified_peptide_label_quant.tsv file:
- No peptides in the output carry the same K with both the heavy label (8.014199) and the digly modification (114.04293) simultaneously.
- The Heavy/Light Match Type columns show a high proportion of unmatched and requantify entries for heavy peptides (Fig. 2, left), unlike a standard SILAC total-proteome search on the same samples not enriched for digly, which shows an even spread of MS/MS, requantify, and unmatched types (Fig. 2, right).
To rule-out a sample-specific issue, I re-analyzed publicly available SILAC-digly data (https://doi.org/10.1016/j.cell.2022.12.025) with the same workflow and observed the same pattern (Fig. 3).
On another attempt, I modified the workflow to include the summed digly+heavy-label mass as an additional variable modification and updated the settings as:
- MSFragger + PTMProphet: added K:122.05713 as a variable mod and in Cmd line opts
- Included detailed mass offsets as: 0.00000(aa=);15.99490(aa=MW);114.04293(aa=K);8.01420(aa=K);122.05713(aa=K);10.00827(aa=R)
- Quant (MS1) labels: Light = K114.04293 and Heavy = K122.05713, in addition to existing labels
This increased MS/MS match types to be more comparable between heavy and light channels (Fig 4, left) and raised the number of modified peptides from 1437 to 2119. However, ratios still do not reverse between the swapped replicates (Fig. 4, right). Log files for both attempts are attached.
Is there a recommended parameter configuration for SILAC-digly searches in FragPipe? Specifically, how should co-modified peptides (digly + heavy SILAC label on the same lysine) be handled in MSFragger, PTMProphet, and in the MS1 quantification settings?
Thank you in advance,
Gabriel
log_files.zip
Hello,
I am attempting a SILAC-digly search by adapting the built-in SILAC3-phospho workflow. My experiment has 2 swap replicates (ctr Heavy/mut Light and mut Heavy/ctr Light), so ratios should reverse between replicates. Instead, both replicates produce ratios in the same direction in combined_modified_peptide_label_quant.tsv (Fig. 1).
Workflow configuration:
I have noticed 2 things in the combined_modified_peptide_label_quant.tsv file:
To rule-out a sample-specific issue, I re-analyzed publicly available SILAC-digly data (https://doi.org/10.1016/j.cell.2022.12.025) with the same workflow and observed the same pattern (Fig. 3).
On another attempt, I modified the workflow to include the summed digly+heavy-label mass as an additional variable modification and updated the settings as:
This increased MS/MS match types to be more comparable between heavy and light channels (Fig 4, left) and raised the number of modified peptides from 1437 to 2119. However, ratios still do not reverse between the swapped replicates (Fig. 4, right). Log files for both attempts are attached.
Is there a recommended parameter configuration for SILAC-digly searches in FragPipe? Specifically, how should co-modified peptides (digly + heavy SILAC label on the same lysine) be handled in MSFragger, PTMProphet, and in the MS1 quantification settings?
Thank you in advance,
Gabriel
log_files.zip