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 Fe3+ 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.