Kinetex evo column

TMT labelled samples were resuspended in 40 µL 200 mM Ammonium formate pH10 and transferred to a glass HPLC vial. BpH-RP fractionation was conducted on an Ultimate 3,000 UHPLC system (Thermo Scientific) equipped with a 2.1 mm ×15 cm, 1.7µ Kinetex EVO column (Phenomenex). Solvent A was 3% ACN, Solvent B was 100% ACN, solvent C was 200 mM ammonium formate (pH 10). Throughout the analysis solvent C was kept at a constant 10%. The flow rate was 500 µL/min and UV was monitored at 280 nm. Samples were loaded in 90% A for 10 min before a gradient elution of 0–10% B over 10 min (curve 3), 10–34% B over 21 min (curve 5), 34–50% B over 5 mins (curve 5) followed by a 10 min wash with 90% B. 15 s (100 µL) fractions were collected throughout the run. Fractions containing peptide (as determined by A280) were recombined across the gradient to preserve orthogonality with on-line low pH RP separation. For example, fractions 1, 25, 49, 73, 97 are combined and dried in a vacuum centrifuge and stored at –20 °C until LC-MS analysis. 24 Fractions were generated in this manner.

For HPLC analysis, 100 µL of the kinase reaction was mixed with 100 µL MeOH and centrifuged at 4 °C and 21.500× g for 20 min. Then, 75 µL of the supernatant were added to 25 µL deionized water, and the mixture was transferred to an HPLC vial with inlet. Samples were analyzed by HPLC-DAD (Agilent 1200 series, Santa Clara/California, USA) at 260 nm using a Kinetex Evo column (C18, 100 A, 250 × 4.6 mm, Phenomenex, Aschaffenburg, Germany) as described previously [12 (link)]. Briefly, a flow rate of 1 mL min⁻¹, a separation temperature of 34 °C, and a gradient of A (KH₂PO₄/K₂HPO₄: 0.1 M, tetrabutylammonium bisulfate: 8 mM, pH 5.4) and B (70% A, 30% methanol) was applied: 0 min—80% A, 4 min—80% A, 14 min—40% A, 35 min—36.5% A, 35.5 min—80% A, and 38 min—80% A.
Typical retention times [min] were for AMP—8.2, ADP—15.4, and ATP—22.9. The retention times of the (deoxy)nucleosides and (deoxy)nucleoside monophosphates are given in Table S5.
The consumed ATP and formed product in the kinase reaction mix were calculated using the following Equations (4) and (5), whereby A_X is the peak area of ATP, A_total is the sum of all adenosine nucleotides (ATP, ADP, AMP), P_X is the peak area of the produced NMP, and P_total is the sum of the substrate and product(s): $$ConsumedAT{P}_{HPLC}[\%]=\left(1-\frac{A_X}{A_{Total}}\right)\times 100$$
$$Produc{t}_{HPLC}[\%]=\frac{P_X}{P_{total}}\times 100$$

TMT labelled samples were resuspended in 40 µL 200 mM ammonium formate pH10 and moved to a glass HPLC vial. BpH-RP fractionation was carried out on an Ultimate 3000 UHPLC system (Thermo Scientific) equipped with a 2.1 mm × 15 cm, 1.7 µm Kinetex EVO column (Phenomenex). Solvent A was 3% acetonitrile, solvent B was 100% acetonitrile, solvent C was 200 mM ammonium formate (pH 10). During the analysis, solvent C was maintained at a constant 10%. The flow rate was 500 µL/min and UV was monitored at 280 nm. Samples were loaded in 90% A for 10 min before a gradient elution of 0–10% B over 10 min (curve 3), 10–34% B over 21 min (curve 5), 34–50% B over 5 min (curve 5) followed by a 10 min wash with 90 % B. 15 s (100 µL) fractions were acquired throughout the run. Fractions that contained peptide (determined by A280) were then recombined across the gradient to preserve orthogonality with on-line low pH RP separation. For example, fractions 1, 25, 49, 73, 97 were combined and dried in a vacuum centrifuge and stored at -20°C until LC-MS analysis. In this manner, 24 fractions were generated.