Guide for Enrichment of Phosphorylated Peptides by Strong Cation Exchange Chromatography
The most commonly used method for the enrichment of phosphorylated peptides is the solid-phase metal ion affinity chromatography (IMAC) described earlier. Although IMAC is relatively simple and fast in application, strong cation exchange chromatography has shown more superior effectiveness in large-scale enrichment experiments. Under the usual strong cation exchange conditions (pH 2.7), most of the peptides produced by trypsin action have two positive charges, and since the phosphate group has one negative charge, the charge of the enzymatically cleaved peptide in the acidic solution is reduced to +1, 0 or lower depending on the phosphate group, and the phosphorylated peptide can be separated and enriched from the multiply charged complex non-phosphorylated peptide sample. However, in practice, because of the complexity of the sample, the selected buffer and separation conditions should also be varied.
This lab manual is designed to help researchers master the basic principles and main steps of strong cation exchange chromatography for the enrichment of phosphorylated proteins and understand the methods and applications of strong cation exchange chromatography for the detection of phosphorylated proteins.
The principle of strong cation exchange chromatography (SCX) for enrichment of phosphorylated peptides is that under normal strong cation exchange conditions (pH 2.7), the degradation of total protein samples by trypsin produces peptides with mostly two positive charges, and since the phosphate group carries a negative charge, the charge of the enzymatically cleaved peptides in acidic solutions is reduced to +1, 0 or lower depending on the number of phosphate groups present. In principle, in strong cation exchange chromatography, the single-charged peptides flow out earlier than the multi-charged peptides, so the phosphorylated peptides can be enriched from the multi-charged complex non-phosphorylated peptides.
The enriched phosphorylated peptides can be analyzed directly by high pressure liquid phase separation and mixed mass spectrometry matrix, or by 2D-PP.
1. Main Instruments and Equipment
Micro-pipette, Proteomelab PF 2D two-dimensional high performance liquid chromatography system, chromatography column (hyper-sil SCX) strong cation exchange column.
2. Material
Total protein separated by various methods.
3. Main Reagents
(1) Trypsin.
(2) Mobile phase A (pH2.7)
| 5mmol/L | NH4CH2PO4 |
| 40% | Acetonitrile |
(3) Mobile phase B*1:350mmol/L NH4Cl
1. 2D Clean-up kit purifies proteins.
2. 2D Quant kit to determine protein concentration.
3. Enzymatic digestion of protein samples.
4. Determination of protein concentration by 2D Quant kit.
5. Dissolve 150μg of the enzymatically digested protein sample in mobile phase A, and inject the sample into high pressure liquid phase separation. The flow rate was 0.7mL/min and the detection wavelength were 214nm. The mobile phase salt concentration gradient was.
100%A，stay for 7min
1 min in 15%B，stay for 5min
10min in 25%B，stay for 5min
10min in 60%B，stay for 5min
1min in 100%B，stay for 10min
1min in 100%A，stay for 10min
Collect one tube of sample every minute with automatic distillate collector
6. Sample detection and analysis
(1) If the protein is not labeled before separation, select the appropriate mobile phase peptide segment for LC separation and mass spectrometry detection.
(2) If the protein has been radiolabeled, 2D-PP detection can be performed.
1. High-purity protein and sufficient enzymatic digestion products can improve the efficiency of enrichment.
2. The concentration of salt ions in the peptide fraction will affect the effect of fractionation, so pay attention to the sample preparation process to minimize the entry of salt ions.
3. The setting of mobile phase salt concentration gradient during fractionation should be adjusted accordingly according to the sample.
*1 Formation of salt gradients with NH4Cl
