An optimized platform for hydrophilic interaction chromatography-immobilized metal affinity chromatography enables deep coverage of the rat liver phosphoproteome.

While analysis of the phosphoproteome has become an important component of understanding how cells function, it remains a nontrivial task in terms of the number of sample preparation steps and instrument time needed to achieve sufficient depth of coverage to produce meaningful results. We previously described a multidimensional method that uses hydrophilic interaction chromatography (HILIC) followed by Fe(3+) immobilized metal affinity chromatography (IMAC) to reduce complexity, improve selectivity, and increase phosphopeptide identifications. Here we present refinements to our overall protocol that make it simpler and more efficient, while they provide greater coverage of the phosphoproteome. We introduce filter-aided sample prep (FASP) for cell lysis and trypsin digestion. Following HILIC separation, fractions are IMAC enriched using a 96-well filter plate. Finally, enriched samples are analyzed using an LC-MS strategy optimized for the fractionation scheme. The optimized protocol improves protein recovery, simplifies phosphopeptide enrichment, and optimizes instrument time, while it maintains deep coverage of the phosphoproteome. By using the refined protocol, we identified more than 16,000 unique phosphosites from rat liver in a single experiment, which used approximately 1 day of instrument time. All together, we present evidence for 24,485 rat liver phosphosites that represents the deepest coverage of a tissue phosphoproteome to date.