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Immunoaffinity precipitation is an effective method of purifying select protein posttranslational modifications (PTMs) for proteomic analysis via mass spectrometry. Peptides containing a modification of interest are isolated directly from protease-digested cellular protein extracts using an antibody with specificity against the modification, and the modified peptides are analyzed by tandem mass spectrometry. Antibodies now exist with specificity for a variety of individual PTMs, such as phosphotyrosine, acetyl-lysine, methyl-arginine, ubiquitylation (i.e., diglycyl-lysine affinity), etc. Here we outline a generalized protocol for the purification of modified peptides by immunoaffinity precipitation. The main restriction for using this protocol is the availability of an antibody against the modification of interest. To purify modified peptides, antibodies are first conjugated to a solid support, such as agarose beads. The beads are then incubated with a complex peptide mixture, derived from a cellular lysate, under neutral pH to facilitate binding of modified peptides. The incubation time can vary from 30 min to overnight, depending upon the antibody used and the complexity of the peptide sample. Finally, acidic buffer conditions are used to elute the PTM-enriched bound peptides for mass spectrometry analysis., (© 2016 Cold Spring Harbor Laboratory Press.)

The addition of posttranslational modifications (PTMs) to proteins is an influential mechanism to temporally control protein function and ultimately regulate entire cellular processes. Most PTMs are present at low stoichiometry and abundance, which limits their detection when analyzing whole cell lysates. PTM purification methods are thus required to comprehensively characterize the presence and dynamics of PTMs using mass spectrometry-based proteomics approaches. Here we describe several of the most influential PTMs and discuss the fundamentals of proteomics experiments and PTM purification methods., (© 2016 Cold Spring Harbor Laboratory Press.)

Immobilized metal affinity chromatography (IMAC) is a frequently used method for the enrichment of phosphorylated peptides from complex, cellular lysate-derived peptide mixtures. Here we outline an IMAC protocol that uses iron-chelated magnetic beads to selectively isolate phosphorylated peptides for mass spectrometry-based proteomic analysis. Under acidic conditions, negatively charged phosphoryl modifications preferentially bind to positively charged metal ions (e.g., Fe(3+), Ga(3+)) on the beads. After washing away nonphosphorylated peptides, a pH shift to basic conditions causes the elution of bound phosphopeptides from the metal ion. Under optimal conditions, very high specificity for phosphopeptides can be achieved., (© 2016 Cold Spring Harbor Laboratory Press.)