Improving the Selectivity of the Phosphoric Acid Βelimination on a Biotinylated Phosphopeptide

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Improving the selectivity of the phosphoric acid βelimination on a biotinylated phosphopeptide

Running title: β-elimination selectivity on a model peptide

Matheron Lucrècea,b, Clavier Séverinea,b, Diebate Omara,b, Karoyan Philippea, Bolbach Gérarda,b, Guianvarc’h Dominiquea, Sachon Emmanuellea,b *.

a Université P. et M. Curie, UMR 7203 UPMC-CNRS-ENS, Laboratoire des Biomolécules, 4, Place Jussieu, 75005 Paris

b Université P. et M. Curie, IFR83, Plateforme de Spectrométrie de Masse et Protéomique, 7-9 Quai Saint Bernard, 75005 Paris, France

- Figure S-1: β-elimination reaction in basic conditions.

- Figure S-2: Reactions observed on the cysteine residues, alkylated or not, during BEM.

- Figure S-3: Kinetic of disappearance of biot-pAdd during BEM in 30% ACN.

- Figure S-4: Kinetic of disappearance of biot-pAdd during BEM in H2O.

- Figure S-5: Kinetic of production of {-H3PO4}biot-pAdd during BEM in 30% ACN.

- Figure S-6: Kinetic of production of {-H3PO4}biot-pAdd during BEM in H2O.

- Table S-1: Attribution of the m/z of the products obtained after BEM on biotpAdd.

Figure S-1: β-elimination reaction in basic conditions. The Ba2+ cations partially neutralize the phosphate group charge, facilitating the attack of the base, and hence catalyze the reaction. X=H (phosphoserine) or CH3 (phosphothreonine).

Figure S-2: Summary of the reactions observed on the cysteine residues after β-elimination alone or after cysteine alkylation followed by β-elimination.

Figure S-3: Comparison of the kinetic of disappearance of the biot-pAdd peptide ([M+H]+ at m/z1759.8) during β-elimination in 30% ACN at 4°C (triangles), room temperature (squares), 37°C (circles) and 50°C (lines). The figure shows the relative areas of the two peaks of interest, extracted from positive ion reflector MALDI-TOF spectrum, with CHCA as a matrix, normalized by the sum of the areas of all the species present on the spectrum. The whole isotopic patterns are taken into account. Number of replicates: n=4 for each measurement.

Figure S-4: Comparison of the kinetic of disappearance of the biot-pAdd peptide ([M+H]+ at m/z1759.8) during β-elimination in H2O at 4°C (triangles), room temperature (squares), 37°C (circles) and 50°C (lines). The figure shows the relative areas of the two peaks of interest, extracted from positive ion reflector MALDI-TOF spectrum, with CHCA as a matrix, normalized by the sum of the areas of all the species present on the spectrum. The whole isotopic patterns are taken into account. Number of replicates: n=4 for each measurement.

Figure S-5: Comparison of the kinetic of production of the {H3PO4}biot-pAdd peptide ([M+H]+ at m/z1661.8) during β-elimination in 30% ACN at 4°C (triangles), room temperature (squares), 37°C (circles) and 50°C (lines). The figure shows the relative areas of the two peaks of interest, extracted from positive ion reflector MALDI-TOF spectrum, with CHCA as a matrix, normalized by the sum of the areas of all the species present on the spectrum. The whole isotopic patterns are taken into account. Number of replicates: n=4 for each measurement.

Figure S-6: Comparison of the kinetic of production of the {H3PO4}biot-pAdd peptide ([M+H]+ at m/z1661.8) during β-elimination in H2O at 4°C (triangles), room temperature (squares), 37°C (circles) and 50°C (lines). The figure shows the relative areas of the two peaks of interest, extracted from positive ion reflector MALDI-TOF spectrum, with CHCA as a matrix, normalized by the sum of the areas of all the species present on the spectrum. The whole isotopic patterns are taken into account. Number of replicates: n=4 for each measurement.

Table S-1: Attribution of the m/z of the products obtained after β-elimination on the biotpAdd peptide. The lines underlined in grey correspond to deletion peptides (synthesis impurities) and to subsequent H2S and/or H3PO4 eliminations. The abbreviation (dhA) stands for dehydroalanine, the residue into which phosphoserine and cysteine residues are converted by BEM. These products dramatically increase with BEM reaction time and are not visible on MALDI-TOF analysis of biotpAdd before β-elimination, indicating that they result from the BEM conditions and not from fragmentation during MALDI-TOF analysis.