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Figure S1. Correct pArg site assignment from MSA-CID and ETD data from in vitro phosphorylation of ClpC by McsB.

Figure S2. Occurrence of dominant neutral loss of H3PO4 upon CID-MS/MS of peptides with pArg

Figure S3. A-D Neutral loss without OH-moieties for neutral loss. Interestingly, intact modification on b-ions is preserved starting from b7, which is the tyrosine residue, that might take part in the neutral loss formation instead of the C-terminus.

Figure S4. High-resolution orbitrap-ETD MS/MS spectrum of PepR_01

Figure S5. Formation of neutral loss species at different activation parameters

Figure S6. CID-MS3 spectra of PepR_04 (pHis) and PepR_05 (pSer) and corresponding y-ion distributions

Figure S7. Ambiguous Phospho-Site Localization of CID-Data

Figure S8 A. Chemically different OH-moieties serve as phospho-acceptors for gas phase rearrangement. Figure S8B CID-MS3-y-ion distribution. Figure S8C Annotated CID-MS3 spectra of peptides PepR_10 to PepR_14

Figure S9. The amidated C-terminus of Pep_11 and Glu12 on Peptide PEP_13 act as phospho-acceptor site under CID conditions.

Figure S10. HCD spectra of PEP_01 and PEP_05

Table TS1. Phosphopeptides of McsB and CtsR resulting from five different enzymatic cleavages.

Table S2. Charge state dependency of phospho-site localization by phosphoRS from CID, MSA-CID, HCD and ETD-MS/MS data for 1% FDR and 2% FDR on the peptide spectrum match level

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