INTRODUCTION
Phosphoproteins and peptides can be bound with high specificity to immobilized metal ions such as Fe3+, Ni2+, and Ga3+. This technique can be used with either on-line or off-line MS analysis. However, elution of the phosphopeptides from the metal ion column prior to MS analysis can result in sample loss. Affinity-bound analytes, including phosphopeptides, can be directly analyzed by MALDI-MS without prior elution from the affinity media. Also, consecutive enzymatic reactions, such as phosphatase or carboxypeptidase Y digestion, can be carried out on affinity-bound peptides. When the affinity-bound phosphopeptides are treated with phosphatase, the number of phosphorylation sites can be determined based on the observation of 80-Da (or multiples of 80 Da) mass shifts in the MALDI-MS of the reaction mixture. Carboxypeptidase Y treatment of the affinity-bound phosphopeptides can also be used to cleave amino acids from the carboxyl terminus, with subsequent direct analysis of the enzymatic products by MALDI-MS to locate the phosphorylation sites on the bound phosphopeptides. This protocol details the preparation and use of Fe3+ or Ga3+ metal IMAC with the on-bead analysis of phosphopeptides by MALDI-MS. Enzymatic digestion of affinity-bound peptides is also described.
MATERIALS
Reagents
Equipment
METHOD
Figure 1. Schematic of affinity binding of phosphopeptides to immobilized metal ion affinity columns.
Affinity Binding of Phosphopeptides to IMAC Column
Alkaline Phosphatase Digestion of Affinity-Bound Phosphopeptides
Carboxypeptidase Y Digestion of Affinity-Bound Phosphopeptides
Perform MALDI analyses on the samples (from Steps 11, 14, and 20) using a delayed-extraction TOF mass spectrometer.
TROUBLESHOOTING
Problem: The CRC leaks during rotation. [Step 9] Solution: Seal the bottom of the CRC with Parafilm. Problem: Peptide detection is below the level of MALDI-MS sensitivity. [Step 21] Solution: MALDI-MS sensitivity is significantly reduced in the presence of glycerol. Ensure that enzyme solutions containing no glycerol. |
Anyone using the procedures in this protocol does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in this protocol and has no liability in connection with the use of these materials. Materials used in this protocol may be considered hazardous and should be used with caution. For a full listing of cautions regarding these material, please consult: Proteins and Proteomics, A Laboratory Manual, by Richard J. Simpson, © 2003 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. 478-481. |
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