The principle of the IMAC protocol is demonstrated by using a semi-complex peptide mixture originating from tryptic digestions of 12 standard proteins (model proteins) that are commercially available. The IMAC purification method is a simple and "easy-to-do" method; however, buffers used for batch incubation with IMAC material should contain 0.1% TFA, 50% acetonitrile to optimize adsorption of phosphopeptides to the IMAC beads, and to reduce nonspecific binding.
1 Model Proteins and Peptide Mixture
a) Dissolve each protein in 50 mM ammonium bicarbonate, pH 7.8, 10 mM DTT and incubated at 37°C for 1 h. After reduction, add 20 mM iodoacetamide and incubate the samples at room temperature for 1 h in the dark. After incubation, quench each reaction with 10 mM DTT.
b) Digest each protein using trypsin (1–2% w/w) at 37°C for 12 h.
2 Batch Incubation with IMAC Beads
a) For 0.3 pmol tryptic digest use 5 μL of IMAC beads. When working with low amounts of sample use less IMAC beads to reduce the level of nonspecific binding from nonphosphorylated peptides. For more complex samples, where more material is available, more IMAC beads should be used.
b) Transfer 5 μL IMAC beads to a fresh 1-mL Eppedorf tube.
c) Wash the IMAC beads twice using 50 μL IMAC loading buffer.
d) Resuspend the beads in 40 μL of IMAC loading buffer and add the sample.
e) Incubate the sample with IMAC beads in a Thermomixer (Eppendorf) for 30 min at 20°C.
3 Packing the IMAC Micro-column
a) Squeeze the tip of a GELoader tip to prevent the IMAC beads from leaking, while still allowing for liquid to pass through.
b) Pack the beads in the constricted end of the GELoader tip by application of air pressure to generate an IMAC micro-column.
c) Wash the IMAC column using 40 μL of IMAC loading buffer.
4 Elution of Phosphorylated Peptides from the IMAC Microcolumn
a) Elute the phosphorylated peptides bound to the IMAC microcolumn using 30 μL of IMAC elution buffer. It is important that this step is performed slowly (∼1 drop/s). (N.B. For MALDI MS analysis the peptides can be eluted off the IMAC micro-column directly onto the MALDI target using 1 μL of DHB solution. After crystallization the sample is ready for MALDI MS analysis).
b) For LC-ESI MSn analysis the IMAC eluate should be acidified using 100% formic acid (pH should be ∼2–3) and desalted/concentrated using reversed phase chromatography (see below).
5 Packing a Reversed Phase (RP) Micro-column for Desalting/Concentrating the Sample
a) Use RP micro-columns of ~3–6 mm (0.4–0.8 μg) depending on the amount of material.
b) Suspend ~2 mg POROS Oligo R3 reversed phase (RP) material in 200 μL of 50% acetonitrile.
c) Squeeze the tip of a GELoader tip to prevent the RP beads from leaking, while still allowing liquid to pass through.
d) Pack the beads in the constricted end of the GELoader tip by application of air pressure forming an micro-column.
6 Loading the Sample Onto a the Reversed Phase (RP) Micro-column
a) Load the acidified sample slowly onto the RP micro-column (~1 drop/sec).
b) Wash the RP micro-column using 30 μL 0.1% TFA.
7 Elution of Phosphorylated Peptides from the RP Micro-column
a) Elute the phosphopeptides from the RP micro-column using 20 μL of RP elution buffer, followed by lyophilization of the phosphopeptides. (N.B. For MALDI MS analysis the peptides can be eluted off the RP micro-column directly onto the MALDI target using 1 μL DHB solution. After crystallization the sample is ready for MALDI MS analysis).
b) LC-ESI-MSn analysis: Redissolve the dried phosphopeptides in 0.5 μL 100% formic acid and immediately dilute to 10 μL with UHQ water.
LC-ESI-MSn analysis of multi-phosphorylated peptides is improved by re-dissolving the phosphopeptides by sonication in an EDTA containing buffer prior to LC-ESI-MS/MS analysis.
- de Graauw, M. (2009). Phospho-Proteomics. Humana Press.