Re-analysis of the data from synthetic peptides run on an orbitrap Fusion (Thermo Scientific) with different acquisition methods (Ferries et al. 2017). The aim is to compare the localisation scores from different bioinformatic pipelines on a low-complexity mix of phospho-peptides with known sequences and phospho-localisation.
The synthetic peptides were spiked in phospho-enriched peptides(Ferries et al. 2017):
Synthetic phosphopeptide standards (∼10 pmol, split into five pools to separate phosphoisomers and thus ensure confidence in phosphosite localization) and 6 μL of enriched phosphopeptides (equivalent to 100 μg of digested cell lysate) were loaded onto the trapping column (PepMap100, C18, 300 μm × 5 mm)I find 179 unique phospho-peptide in the pools. This is not in line with what is described in (Ferries et al. 2017).
Here is the head of the corresponding table:
| Phospopeptide.sequence | IPI.acc..No. | Protein | X..of.sites.in.peptide | modified.position.in.peptide | Neutral.mass.SH.Cys | position.in.96.well.plate | pool | Pool | PhosphopeptideID | PhosphosequenceID |
|---|---|---|---|---|---|---|---|---|---|---|
| ADENYYK | IPI00018597 | SYK | 1 | 5 | 981.3481 | plate 1 /well A 1 | 1 | pool_1 | pool_1_ADENYYK_5 | pool_1_ADENYYK_1 |
| AGGKPSQSPSQEAAGEAVLGAK | IPI00216969 | ABL1 | 1 | 6 | 2118.9946 | plate 1 /well B 7 | 1 | pool_1 | pool_1_AGGKPSQSPSQEAAGEAVLGAK_6 | pool_1_AGGKPSQSPSQEAAGEAVLGAK_1 |
| AVGMPSPVSPK | IPI00004344 | AFF4 | 1 | 9 | 1148.5300 | plate 1 /well C 1 | 1 | pool_1 | pool_1_AVGMPSPVSPK_9 | pool_1_AVGMPSPVSPK_1 |
| DKSPSSLLEDAK | IPI00329488 | ABL2 | 1 | 3 | 1368.6173 | plate 1 /well D 1 | 1 | pool_1 | pool_1_DKSPSSLLEDAK_3 | pool_1_DKSPSSLLEDAK_1 |
| ESKSSPRPTAEK | IPI00004344 | AFF4 | 1 | 4 | 1395.6395 | plate 1 /well E 1 | 1 | pool_1 | pool_1_ESKSSPRPTAEK_4 | pool_1_ESKSSPRPTAEK_1 |
| FGESDTENQNNK | IPI00328149 | EIF2AK1 | 1 | 4 | 1461.5409 | plate 1 /well F 1 | 1 | pool_1 | pool_1_FGESDTENQNNK_4 | pool_1_FGESDTENQNNK_1 |
I use the field PhosphopeptideID to check the correct/incorrect identification and localisation. The field PhosphosequenceID informs on the sequence and number of phosphorylation, but not on their localisation. I use it to match the PSMs from specific pools to the correct/expected phosphorylation localisation.
I identify the isomers as the peptide sequences that can present several combinations of phospho-localisation.
| Var1 | Freq |
|---|---|
| 1 | 98 |
| 2 | 28 |
| 3 | 4 |
| 4 | 2 |
| 5 | 1 |
Venn diagram of the sequences in each pool (phospho-isomers are present in several pools):
| pool_1 | pool_2 | pool_3 | pool_4 | pool_5 | |
|---|---|---|---|---|---|
| 2 | 5 | 3 | 0 | 5 | 3 |
| 1 | 31 | 33 | 36 | 31 | 32 |
Ferries, Samantha, Simon Perkins, Philip J. Brownridge, Amy Campbell, Patrick A. Eyers, Andrew R. Jones, and Claire E. Eyers. 2017. “Evaluation of Parameters for Confident Phosphorylation Site Localization Using an Orbitrap Fusion Tribrid Mass Spectrometer.” Journal of Proteome Research 16 (9): 3448–59. https://doi.org/10.1021/acs.jproteome.7b00337.