Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides

Abstract

In contrast to protein O-phosphorylation, studying the function of the less frequent N- and S-phosphorylation events have lagged behind because they have chemical features that prevent their manipulation through standard synthetic and analytical methods. Here we report on the development of a chemoselective synthetic method to phosphorylate Cys side-chains in unprotected peptides. This approach makes use of a reaction between nucleophilic phosphites and electrophilic disulfides accessible by standard methods. We achieve the stereochemically defined phosphorylation of a Cys residue and verify the modification using electron-transfer higher-energy dissociation (EThcD) mass spectrometry. To demonstrate the use of the approach in resolving biological questions, we identify an endogenous Cys phosphorylation site in IICB(Glc), which is known to be involved in the carbohydrate uptake from the bacterial phosphotransferase system (PTS). This new chemical and analytical approach finally allows further investigating the functions and significance of Cys phosphorylation in a wide range of crucial cellular processes.

Figure 1. Synthetic strategy to install pCys residues in unprotected peptides.

Site-selective addition of phosphite to an activated Ellman-disulfide to deliver a stereochemically defined pCys residue.

Figure 2. Chemoselective synthesis of phosphorothiolate esters peptides 4 and 5.

(a) Reaction of peptide 1a with Ellman's reagent and peptide 2a with 5 eq. phosphite esters 3a–e. (b) Overlap of UPLC traces of reaction mixture for peptide 2a and phosphite 3d after 16 h (black) and phosphite 3d starting material (magenta). Formation of the phosphorothiolate ester 4d after 16 h incubation with phosphite 3d in DMF at room temperature. Magenta asterisk shows the by-products of the phosphite 3d decomposition, which overlap with the reaction mixture UPLC trace for peptide 2a and phosphite 3d. (c) UPLC-UV trace after purification by semi-preparative HPLC of phosphorothiolate ester 4d. a.u., arbitrary unit; r.t., room temperature.

Figure 3. Ultraviolet-deprotection and analytical characterization of pCys peptide 6a and 6b.

(a) Light-induced photolysis of peptide 4d to deliver pCys peptide 6a in 65% isolated yield. (b) UPLC-UV traces before (in blue) and directly after ultraviolet light exposure (in magenta). (c) ³¹P-NMR experiment showing the characteristic peak for pCys residues (12.02 p.p.m.) and inorganic phosphate. (d) EThcD MS/MS spectra of pCys peptide 6b, showing complete sequence coverage and diagnostic fragment ions c4, and z10. a.u., arbitrary unit.

Figure 4. Characterization of an endogenous pCys peptide by EThcD MS/MS.

(a) Glucose-specific phosphoenolpyruvate-dependent phosphotransferase system. The phosphate group is transfered from PEP sequentially to the IIB^Glc domain of the membrane protein IICB^Glc. (b) SDS–PAGE in-gel trypsin digeston approach. (c) In solution tryptic digestion and offline two-dimensional LC approach.

Figure 5. Analysis by nLC-ESI-EThcD MS/MS of the endogenous pCys peptide.

(a) Total ion chromatogram (TIC) of the tryptic digestion after in vitro phosphorylation. (b) Extracted ion chromatogram (XIC) of the phosphorylated Cys peptide with m/z 721.821. (c) EThcD MS/MS spectra of the endogenous pCys peptide, ENITNLDApCITR, showing complete sequence coverage and diagnostic fragment ions c9, y4 and z5.