RPC was performed on a Thermo EASY nano-LC 1000 system
(Thermo Fisher Scientific) equipped with a PicoFrit PLRP-S column
(100 mm × 100 μm i.d., 5 μm, 1000 Å; New Objective,
Inc., Woburn, MA). The following buffers were used for RPC: buffer
A, water with 0.25% formic acid; buffer B, acetonitrile with 0.25%
formic acid. The nano-LC was operated at a flow rate of 500 nL/min,
and 1 μL of sample was injected with an autosampler after equilibration
of the capillary column. For the separation of both standard proteins
and E. coli cell lysate proteins, an
80
min optimized RPC gradient was used consisting of the following concentrations
of buffer B: 5% for 15 min, 25% at 25 min, 60% at 70 min, 95% at 75
min, and then back to 5% at 80 min. Prior to injection on the RPC
column, a brief desalting procedure (three times with 10 kDa ultracentrifugal
filters) was performed on both the E. coli cell lysate fractions after HIC with ammonium tartrate salt gradient
and standard proteins prepared in 1.8 M ammonium tartrate buffer to
remove a substantial amount of salt.
(Thermo Fisher Scientific) equipped with a PicoFrit PLRP-S column
(100 mm × 100 μm i.d., 5 μm, 1000 Å; New Objective,
Inc., Woburn, MA). The following buffers were used for RPC: buffer
A, water with 0.25% formic acid; buffer B, acetonitrile with 0.25%
formic acid. The nano-LC was operated at a flow rate of 500 nL/min,
and 1 μL of sample was injected with an autosampler after equilibration
of the capillary column. For the separation of both standard proteins
and E. coli cell lysate proteins, an
80
min optimized RPC gradient was used consisting of the following concentrations
of buffer B: 5% for 15 min, 25% at 25 min, 60% at 70 min, 95% at 75
min, and then back to 5% at 80 min. Prior to injection on the RPC
column, a brief desalting procedure (three times with 10 kDa ultracentrifugal
filters) was performed on both the E. coli cell lysate fractions after HIC with ammonium tartrate salt gradient
and standard proteins prepared in 1.8 M ammonium tartrate buffer to
remove a substantial amount of salt.