• Characterization of Phosphopeptides Using a Combination of Immobilized Metal Ion

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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

Acetic acid (100 mM)
 

Ammonium bicarbonate (50 mM, pH 8.0)
 

Calf intestinal alkaline phosphatase
 

Carboxypeptidase Y (1 µg/µL in H2O)
 

EDTA (100 mM in H2O, pH 8.0)
 

Ferric chloride (FeCl3) (100 mM)
 

Gallium chloride (GaCl3) (60 mM)
 

MALDI matrix A Prepare a saturated solution of {alpha}-cyano-4-hydroxy-cinnamic acid in:
 

• Ethanol (45% [v/v])
• H2O (45% [v/v])
• 10% formic acid (10% [v/v]).
• Store in the dark at room temperature.
   
  

Nickel-nitrilotriacetic acid (Ni-NTA) resin
 

Phosphoprotein of interest
 

Sodium citrate (50 mM, pH 6.0)
 

TPCK-modified trypsin (sequence-grade)

Equipment

Compact reaction columns (CRC) and filters (35-µm pore size)
 

Incubator (slow rotation, e.g., rotating wheel)
 

MALDI mass spectrometer (e.g., the Voyager DE-STR) or equivalent
 
The Voyager DE-STR mass spectrometer (Applied Biosystems) is equipped with a nitrogen laser (337-nm) to desorb and ionize the samples. A close external calibration, using two points to bracket the mass range of interest, should be performed prior to analyzing the protein sample.
 

MALDI target
 

Microcentrifuge
 

Microcentrifuge tubes (standard and 0.7-mL)

METHOD

Figure 1. Schematic of affinity binding of phosphopeptides to immobilized metal ion affinity columns.