Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Phosphorylated Amino Acids
Methods of Analysis

8.11.2.1. Thin-Layer Chromatography.

1. On MN-300 Cellulose plates. Eluent: butanol — isopropanol — formic acid — Water (3:1:1:1) ([358], details not given).

2. On polyamide plates (Cheng-Chin). Chromatograph in one direction using 5% propionic acid containing 0.013–0.025% SDS. Dry, spray with a 0.2% ninhydrin solution, and heat in an oven at 50 °C while monitoring. Thr(P) and Ser(P) migrate together, R_f = 0.70; Tyr(P) = 0.54.

8.11.2.2. Electrophoresis [42, 169]. pH 1.9 buffer: glacial acetic acid — formic acid (88%) — water (78:25:897); pH 3.5 buffer: glacial acetic acid — pyridine — water (50:5:945). Pyridine is purified by refluxing over p-toluenesulfonyl chloride for 18 h and then distilled.

Use thin-layer plates (Brinkman, Polygram CEL MN-300, 200X200 mm, 0.1 mm). Dissolve the sample in pH 1.9 buffer and perform electrophoresis at pH 1.9, 1.5 kV, for 75 min toward the anode. After drying, perform electrophoresis In the second direction using pH 3.5 buffer, 1 kV, for 1 h. Dry the plate, spray with a buffer solution containing 0.1% ninhydrin (Section 8.14.2). Tyr(P) gives a gray color; Ser(P) and Thr(P) give a blue color. For radioactive samples, use autoradiography.

Standard solutions: a mixture of 0.5 µL each of neutralized phosphoamino acid solutions (20 mg/mL). Fig. 8.2 shows the positions of the phosphoamino acid spots after two-dimensional electrophoresis.

8.11.2.3. Amino acid analyzer [51]. Use a short Column (according to Moore and Stein) (80X6.0 mm) packed with Durrum DC6A resin. Dissolve the sample in 0.01 M TFA with or without unlabeled phosphoamino acid standards (1.5 nmol each) and load onto the column. Isocratic elution is performed at 50 °C with 0.01 M TFA at a flow rate of 0.4 mL/min. For fluorimetric detection, the fractions are treated with OPA reagent (Section 8.16.3.2) (flow rate 0.2 mL/min) in a special mixer. When working with a [³²P]-labeled sample, replace the OPA reagent with water and collect 1 mL fractions to measure radioactivity.

Approximate retention times: Ser(P) 19 min, Thr(P) 28 min, Tyr(P) 64 min. Cysteic acid elutes in the void volume, and Other Amino Acids do not elute.

Regenerate the column by passing the following solutions through it:

1) 1.0 M in Na⁺ (34.8 g NaCl + 39.2 g trisodium citrate dihydrate per 1 L of water with The addition of 1 mL of phenol to inhibit bacterial growth);

2) 0.2 M NaOH;

3) 1 M TFA;

4) 0.01 M TFA.

Quantitative analysis results. The yields for standard phosphoamino acid samples (determined by fluorescence measurement) were: Ser(P) 88%, Tyr(P) 71% relative to the yield of Thr(P) taken as 100%, and, respectively, Thr(P) 89%, Ser(P) 71%, and Tyr(P) 71% relative to the yields of their non-phosphorylated analogs.

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FIG. 8.2. Two-dimensional electrophoretic Separation of phosphoamino acids: in the first direction at pH 1.9, in the second direction at pH 3.5 [43].

8.11.2.4. High-pressure liquid chromatography.

1. Anion-exchange column (Ultrasil AX, 250X4.6 mm, Beckman) equilibrated with 15 mM potassium phosphate buffer (pH 3.8). Apply the sample and elute with the same buffer for 25 min at a flow rate of 2.0 mL/min. Non-radioactive compounds are detected by measuring absorbance at 206 nm. Approximate retention times: Thr(P) 6 min; Ser(P) 8.6 min; Tyr(P) 9 min; and P_i 10.5 min [358].

2. Anion-exchange column (SynChropak AX-300, 250X4.6 mm). Elute with phosphate buffer (as in step 1) at a flow rate of 1 mL/min. The eluate fractions are treated with OPA [213], and absorbance is measured at 338 nm, or radioactivity is determined using a liquid scintillation counter. Retention times: Thr(P) 12 min; Ser(P) 17 min; Tyr(P) 23 min (J. Downward, personal communication).

3. Column (250X4.6 mm) packed with Partisil-10 SAX anion-exchange resin (Whatman). Apply the sample and elute with 10 mM potassium phosphate buffer (pH 3.0) containing 10.5% methanol at a flow rate of 1 mL/min at room Temperature. Detection is performed using OPA derivatization. Approximate retention times: Thr(P) 26 min; Tyr(P) 32 min; Ser(P) 38 min; P_i 60 min [413].

4. Dansylation and HPLC of DNS-phosphoamino acids [77]. Hydrolyze the protein for 2 h at 110 °C with concentrated Hydrochloric acid (Pierce) and evaporate to dryness under a stream of nitrogen. Add 100 µL of 0.2 M Na₂CO₃ and evaporate again. Suspend the sample in 100 µL of water and add 100 µL of freshly prepared dansyl chloride solution (2.5 mg DNS-Cl per 1 mL of acetone). Incubate in the dark for 1 h at 37 °C. Dry thoroughly under a stream of nitrogen. For HPLC analysis, dissolve the DNS derivatives in 100 µL of 0.5% TFA.

HPLC on a Protesil 300 column (Whatman). Wash the column with water — TFA (1000:1) or water — acetonitrile — TFA (970:30:1). Elute using a linear gradient: 30 mL of water — acetonitrile — TFA (970:30:1) + 30 mL of water — acetonitrile — TFA (900:100:1). Measure the absorbance of the eluate at 280 nm. Collect radioactive fractions for counting. Approximate retention times: Ser(P) 12 min, Thr(P) 16 min, and Tyr(P) 22 min.

HPLC on a Bondapak C₁₈ microcolumn (Waters). Isocratic elution with water — acetonitrile — TFA (925:75:1). Approximate retention times: Ser(P) 15 min, Thr(P) 22 min; Tyr(P) was not detected in the eluate.

5. Ion-pair reversed-phase HPLC of phosphoamino acids [1, 268]. Column (150X4.6 mm) with Partisil 5 ODS2. Elute at room temperature with a mixture of 0.5 mM tetrabutylammonium hydroxide — 0.5 mM o-phthalate, pH 6.3, at a flow rate of 1 mL/min. Detection at 243 nm, scale 0.1 AUFS, sensitivity 200 pmol. Approximate retention times: Asp and Glu 5.3 min; P_i 6.9 min; Thr(P) 11.6 min; Ser(P) 12.8 min; Tyr(P) 15.9 min.