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. 2019 Sep 10;8:e46490. doi: 10.7554/eLife.46490

Table 3. Analysis of the cellular proteome for oxidised peptides encompassing reported sites of prolyl hydroxylation.

The number of unmodified and singly oxidised peptides containing reported sites of PHD-catalysed hydroxylation were counted over a range of stringency filters. Where multiple reported oxidation sites of one protein occur on separate peptides the number of spectra recorded is a summation of all interrogated peptides. The number of assigned target site prolyl oxidations is indicated in bold; methionine (Met) and alternate oxidation sites, including non-target proline residues (Other) are also presented together with unoxidised peptides for comparison. Stringency filters were applied as follows; PTM assignment confidence as assessed by ambiguity score (AScore:>20) and fragment ion intensity (Ion Intensity:>5%). These filters were applied separately and in combination to derive a list of assigned peptides with confidently localised oxidations. There are no high confidence proline oxidation assignments (i.e., meeting both AScore and Ion Intensity thresholds) of the reported substrates, suggesting a high degree of uncertainty from the software.

Table 3—source data 1. Summary of non-enzymatic oxidation assignments on synthetic peptide standards.
MSMS assignment frequency of artefactual oxidations observed on unmodified tryptic peptide standards; oxidations are stratified by residue (e.g., Met, Pro, other). Reported assignments (column 4) were not subject to PTM localisation (AScore) filtering. Oxidations assigned to target Pro residues are indicated in red. Variation in the total number of peptide assignments (Column 5) reflects differences in the amount of peptide injected and/or replicate runs.
elife-46490-table3-data1.docx (34.1KB, docx)
DOI: 10.7554/eLife.46490.053
Table 3—source data 2. Peptide identification results from database search represented in Table 3.
elife-46490-table3-data2.xlsx (245.8KB, xlsx)
DOI: 10.7554/eLife.46490.054

 # Spectra filtered by confidence of modification site
Gene ID Reported site Assigned No filter AScore Ion Intensity AScore and Ion Intensity
ACACB P343, P450, P2131 Unoxidised 63 N/A N/A N/A
ACTB P307, P322 P322 57 7 11 -
Met 4528 647 4096 406
Other 336 58 62 7
Unoxidised 8650 N/A N/A N/A
ADRB2 P382*, P395* Unoxidised 1 N/A N/A N/A
AKT1 P125, P313*, P318*, P423 Unoxidised 160 N/A N/A N/A
CENPN P311 Unoxidised 7 N/A N/A N/A
EEF2K P98 Met 5 - - -
Unoxidised 41 N/A N/A N/A
FLNA P2317*, P2324* P2324 2 - - -
Other 1 - - -
Unoxidised 562 N/A N/A N/A
FOXO3 P426*, P437* Unoxidised 4 N/A N/A N/A
NDRG3 P294 Met 13 5 - -
Unoxidised 49 N/A N/A N/A
PDE4D P29, P382, P419 Met 14 13 9 9
Other 1 - - -
Unoxidised 23 N/A N/A N/A
PKM P403*, P408* P403 3 1 - -
P408 5 - - -
Other 5 - - -
Unoxidised 3394 N/A N/A N/A
PPP2R2A P319 Met 50 11 26 11
Other 18 - - -
Unoxidised 119 N/A N/A N/A
SPRY2 P18, P144, P160 Unoxidised 7 N/A N/A N/A
TELO2 P374, P419, P422 Unoxidised 47 N/A N/A N/A
THRA P160*, P162* Unoxidised 20 N/A N/A N/A
TP53 P142, P359 Unoxidised 7 N/A N/A N/A









Total Pro 67 8 11 0
Met 4610 676 4131 426
Other 361 58 62 7
Unoxidised 13154 0 0 0
All 18192 742 4204 433

*Doubly oxidised peptides were interrogated when multiple prolyl hydroxylation sites had been reported on the same tryptic peptide.