Tskgel amide 80 5 μm particle column

Proteolytic digestion products were desalted on a 1-g Sep-Pak cartridge (Waters, Milford, MA), and subjected to hydrophilic interaction liquid chromatography (HILIC) using a 4.6 × 250-mm TSKgel Amide-80 5-μm particle column (Tosoh Biosciences, Tokyo, Japan). The following buffers were used: HILIC buffer A (0.1% TFA) and HILIC buffer B (99.9% acetonitrile and 0.1% TFA). The peptide pellets were resuspended in 80% HILIC buffer B and injected onto the HILIC column. The chromatography was done using the following elution gradient: 80% B held for 20 min followed by 80% B to 60% B in 40 min and finally 0% B for 10 min.
Eighteen fractions of each subcellular compartment were collected throughout the gradient and further enriched in phosphopeptides by immobilized metal affinity chromatography (IMAC, Phos-Select, Sigma) following manufacturer's instructions. Peptides were eluted with 200 μl, 0.4 m NH4OH followed with 200 μl, 0.2 m NH4OH/50% acetonitrile. A gel-loading tip was used to remove elution fractions and further remaining IMAC beads were removed by passing fractions through a ZipTip (Millipore). The supernatants from the IMAC (combining the 18 fractions for each subcellular compartment in nine samples) were used for additional TiO₂ pull down (Titanspheres, GL Sciences, Shinjuku, Japan).

The peptides
were fractionated using hydrophilic interaction liquid chromatography
(HILIC) according to a previously published protocol.^{18 (link)} All HILIC experiments were performed on an Agilent 1100
HPLC system using a 1 × 250 mm TSKgel Amide-80 5 μm particle
column (Tosoh Biosciences). Peptides were loaded in 90% solvent B
(98% acetonitrile with 0.1% TFA). Solvent A consisted of 2% acetonitrile
with 0.1% TFA. Peptides were eluted with an inverse gradient of 90%
B to 85% B in 5 min followed by 85% B to 70% B in 40 min and finally
a steep gradient to 0% B in 5 min at 0.05 mL/min. Twelve fractions
were collected from HILIC separation and analyzed by LC–MS.
Ten of these fractions contained peptides.