The fractions were sequentially loaded onto a
μ-precolumnTMcartridge (0.3 × 5 mm2, 5 μm; Dionex) at a
flow rate of 20 μL/min. The trap column effluent was
then transferred to a reverse-phase microcapillary column
(Acclaim® PepMap100 C18 column, 0.075 × 150 mm2, 3 μm,
100 A; Dionex). Reverse-phase separation of peptides was
performed using the following buffers: buffer A (2% ACN and
0.5% acetic acid) and buffer B (80% ACN and 0.5% acetic acid); a
120-min gradient (4% to 7% of buffer B for 3 min, 7% to 33% of
buffer B for 102 min, 33% to 50% of buffer B for 10 min, 50% to
100% of buffer B for 1 min, 100% of buffer B for 3 min, and
100% to 4% buffer B for 1 min) was used