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
- Ethanol (45% [v/v])
- H2O (45% [v/v])
- 10% formic acid (10% [v/v]).
- Store in the dark at room temperature.
Equipment
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.
METHOD
Figure 1. Schematic of affinity binding of phosphopeptides to immobilized metal ion affinity columns.
- Insert the CRC filter (35-µm) into the CRC. Place the CRC in a standard microcentrifuge tube.
- Add ~30 µL of the Ni-NTA resin slurry to the bottom of the CRC tube. Drain the column by centrifugation at 120g for 1 min at room temperature.
These centrifugation conditions are the same for all washes unless otherwise stated. - Wash the Ni-NTA resin three times each with 30 µL of 100 mM EDTA (pH 8.0) to remove the bound Ni2+ metal.
- Wash the column three times each with 30 µL of H2O.
- Wash the column three times each with 30 µL of 100 mM acetic acid.
- To regenerate the columns with either Fe3+ or Ga3+ metal, wash the column three times each with 30 µL of either 60 mM GaCl3 or 100 mM FeCl3.
- To remove any unbound metal ions, wash the column three times each with 30 µL of H2O followed by three times each with 30 µL of 100 mM acetic acid.
-
Affinity Binding of Phosphopeptides to IMAC Column
- Digest the phosphoprotein (~0.1 µg/µL) with trypsin (protein:enzyme ratio of 20:1 to 100:1 [w/w]) in 50 mM NH4HCO3 buffer (pH 8) for 2 h at 37°C.
- Load 20 µL (~20 µg) of the trypsin-digested phosphorylated protein with 30 µL of 100 mM acetic acid onto the IMAC column. Incubate the column on a slowly rotating wheel for 30 min at 37°C.
Check the column periodically for leakage from the bottom of the CRC. See Troubleshooting.
- Remove unbound peptides from the IMAC column by washing the column with 30 µL of H2O followed by 30 µL of 100 mM acetic acid. Repeat this step three times. The IMAC column (with bound phosphopeptides) can be stored in 100 mM acetic acid for at least 2-3 d at 4°C. Even bound phosphopeptides from a complex mixture such as a cell extract should be stable because endogenous phosphatases are digested by the trypsin and/or elute from the column. To verify that nonspecific peptides are washed off the column or if the unbound peptides are of interest, analyze the washes by MALDI-MS.
- Spot a 0.5-µL aliquot of settled beads with 0.5 µL of MALDI matrix on the MALDI target
Alkaline Phosphatase Digestion of Affinity-Bound Phosphopeptides
- Mix a 5-µL aliquot of the phosphopeptides affinity-bound to the IMAC media (from Step 10) with 5 µL of 50 mM NH4HCO3 (pH 7.8) and 1 unit of calf intestine alkaline phosphatase in a 0.7-mL microcentrifuge tube.
- Incubate the tube on a slowly rotating wheel at 37°C.
- Monitor the time course of the digestion periodically by spotting a 0.5-µL aliquot of the reaction mixture (supernatant and beads) with 0.5 µL of MALDI matrix onto a MALDI target. Take the first aliquot 30 min after beginning Step 13.
Initially, additional aliquots are typically monitored at 30-min intervals. Depending on the protein, incubation times for the digestion can range from 1 h to overnight.
Carboxypeptidase Y Digestion of Affinity-Bound Phosphopeptides
- Transfer a 25-µL aliquot of the phosphopeptides affinity-bound to the IMAC media (from Step 10) into another CRC.
- Wash the column three times each with 30 µL of 50 mM sodium citrate (pH 6).
- Add 40 µL of 50 mM sodium citrate (pH 6) to the column.
- Add 1 µL of carboxypeptidase Y solution to the column.
- Incubate the column on a slowly rotating wheel at 37°C.
- Depending on the protein, the carboxypeptidase Y digests can take from a few minutes to overnight to complete. Thus, the time course of the digest should be monitored initially at 30- to 60-sec intervals. To monitor the time course of the reaction:
- the beads three times each with 50 µL of 100 mM acetic acid.
- Add 30 µL of 100 mM acetic acid.
- Spot 0.5 µL of beads with 0.5 µl of MALDI matrix onto the MALDI target for MALDI-MS analysis.
- Resume the carboxypeptidase Y reaction by repeating Steps 16-19.
Mass SpectrometryPerform MALDI analyses on the samples (from Steps 11, 14, and 20) using a delayed-extraction TOF mass spectrometer.
TROUBLESHOOTINGProblem: 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.
- Digest the phosphoprotein (~0.1 µg/µL) with trypsin (protein:enzyme ratio of 20:1 to 100:1 [w/w]) in 50 mM NH4HCO3 buffer (pH 8) for 2 h at 37°C.
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