1 Enrichment of Phosphopeptides Before MALDI-TOF and Nanoelectrospray MS Using Metal-Affinity Chromatography Pipet Tips
1.1 Charging and Equilibration of the ZipTipMC
a) Prepare 1 mL metal ion solution.
b) Wash tip and dispense to waste three times with 10 μL fresh 0.1% acetic acid with 50% acetonitrile.
c) Load the column with metal ions by charging the tip with multiple column volumes of this solution by carrying out 10 aspirate and dispense cycles with 10-μL aliquots of metal ion solution.
d) Wash the tip and dispense to waste three times with 10 μL of high-purity water. Wash the tip and dispense to waste three times with 10 μL of fresh 1.0% acetic acid, 10% acetonitrile.
e) Equilibrate the tip by washing and dispensing to waste five times with 10 μL of appropriate binding solution.
f) After use, the tips may be washed and regenerated for reuse up to three times.
1.2 Binding and Washing Using MES Buffer (pH 5.5)
a) Dilute sample in acidic binding solution, such as MES, formic acid, or acetic acid in aqueous acetonitrile.
b) Bind sample to ZipTip by fully depressing the pipet plunger to a dead stop using a 1-to 10-μL volume.
c) Aspirate and dispense the sample 5 to 10 cycles for maximum binding.
d) Wash tip and dispense to waste three times with 10 μL of binding solution.
e) Wash tip and dispense to waste three times with 10 μL 0.1% acetic acid with 50% acetonitrile.
f) Wash tip and dispense to waste three times with 10 μL high-purity water.
1.3 Alterative: Binding and Washing Using 0.1% Acetic or 0.01% Formic Acid
a) Dilute the sample in binding solution (0.1% acetic or 0.01% formic acid).
b) Bind the sample to the ZipTip by fully depressing the pipet plunger to a dead stop using a 1- to 10-μL volume.
c) Aspirate and dispense the sample 5 to 10 cycles for maximum binding.
d) Wash tip and dispense to waste three times with binding solution.
e) Wash tip and dispense to waste three times with 10 mL 0.1% acetic acid (or 0.01% formic acid), 50% acetonitrile.
f) Wash tip and dispense to waste three times with high-purity water.
1.4 Eluting the Phosphopeptides for MALDI-TOF MS
a) Pipet 2 μL of freshly prepared ammonium hydroxide elution solution into a clean vial using a standard pipet tip.
b) Aspirate and dispense eluant through the ZipTip four to six times without introducing air.
c) For direct spotting onto a MALDI-TOF MS target plate, aspirate the desired volume of eluted phosphopeptide solution into the ZipTip and dispense directly onto target, let dry partially, then overspot with 1 μL of matrix.
1.5 Eluting the Phosphopeptides for Nanoelectrospray MS (see Note 6)
a) Carry out steps 1 and 2 in Subheading 1.4.
b) For direct loading into a nanoelectrospray MS needle, elute the sample into a clean Eppendorf tube or vial, or use a GELoader tip to introduce directly into a nanospray needle.
c) Cut the GELoader tip approx 2–3 mm above where the tip is fused to its capillary (i.e., narrow) end.
d) Before final dispensing of the sample, press the cut-down GELoader tip firmly onto the ZipTip pipet tip with a slight twisting motion. The leak-free fit allows elution directly into the nanospray needle.
2 Modification of Phosphoserine to S-Ethylcysteine
a) In a fume cupboard, the peptide is dissolved in a capped tube containing 50 μL incubation mixture in a capped Eppendorf tube.
b) Flush the tube with nitrogen and incubate for 1 h at 50°C.
c) After allowing to cool, add 10 μL of glacial acetic acid.
d) Apply the derivatized peptide either directly for analysis or concentrate first by vacuum centrifugation.
e) The β-elimination step during derivatization of a phosphoserine adjacent to a proline residue is slow; therefore, reaction time may be extended to 18 h at 50°C. Acetonitrile can be used instead of the normal solvents to minimize subsequent manipulations.
f) This procedure is also used for the selective isolation of phosphoseryl peptides. When the S-ethylcysteinyl peptides are applied to a reverse-phase HPLC column (e.g., Vydac C18) and eluted with linear gradients of water/acetonitrile in 0.1% trifluoroacetic acid (TFA), the derivatized peptides emerge on average 4 to 5% acetonitrile later than the native phosphopeptide. A derivatized peptide from a doubly phosphorylated species will elute correspondingly later than the singly derivatized species, indicating the applicability of the method to multiple-phosphoseryl peptides. HPLC before and after derivatization by this "diagonal" technique should produce highly purified peptides even from a very complex mixture, since the elution position of all others will be unaffected.
3 Selective Detection of Phosphopeptides on Mass Spectrometry
a) Phosphate-specific fragment ions of 63 Da (PO2-) and 79 Da (PO3-) are produced by collision-induced dissociation during negative-ion LC-electrospray MS. This technique of selective detection of posttranslational modifications through collision-induced formation of low-mass fragment ions that serve as characteristic and selective markers for the modification of interest, has been extended to identify other modifications such as glycosylation, sulphation, and acylation.
b) Ladder sequencing by MS involves the generation of a set of nested fragments of a polypeptide chain followed by analysis of the mass of each component. Each component in the ragged polypeptide mixture differs from the next by loss of a mass that is characteristic of the residue weight (which may involve a modified side chain). In this manner, the sequence of the polypeptide can be read from the masses obtained in MS.
4 Detection of Phosphopeptides After Dephosphorylation With Alkaline Phosphatase
a) Carry out MALDI-TOF mass analysis of aliquots of each sample that has been enriched in phosphopeptides after IMAC or after HPLC.
b) Dissolve separate samples in 0.5 μL of 50mM NH4HCO3, pH 8.9, containing 0.05 or 1 unit of calf intestinal alkaline phosphatase/μL.
c) Incubate samples for 2 h at 37°C.
d) For MALDI-TOF, the peptides may be desalted on C18 ZipTips and eluted directly onto the target plate with 80% (v/v) aqueous acetonitrile, 0.1% TFA, containing the MALDI matrix, α-cyano-4-hydroxycinnamic acid (Aldrich).
e) A mass shift of 80 Da caused by the removal of the phosphate allows unambiguous determination of whether a particular peptide is phosphorylated.
4.1. On-Target Alkaline Phosphatase Treatment
a) Carry out MALDI-TOF mass analysis of samples enriched in phosphopeptides as in Subheading 3.4. using α-cyano-4-hydroxycinnaminic acid matrix.
b) Dissolve the sample/matrix mixture on the target with 1–1.5 μL of 50 mM ammonium bicarbonate containing 0.05 U/μL alkaline phosphatase.
c) Incubate the same samples in situ with the alkaline phosphatase on the MALDI target for 1–2 h at 37°C in a closed high-humidity chamber (a plastic box with a snap-on lid, containing wet tissue, is ideal) to prevent drying.
d) Stop the dephosphorylation reaction by the addition of 0.5 μL of acetonitrile/TFA solution and dry samples immediately in vacuo to allow proper crystallization of the matrix.
e) Repeat the MALDI-TOF MS as before to determine which peptides have altered in mass by 80 Da (or multiples thereof).
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Reference
- Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.
