Half of the TMT-labeled peptides mixture was fractionated using a Waters XBridge BEH130 C18 3.5 μm 2.1 × 150 mm column on an Agilent 1290 HPLC operating at 0.2 mL/min. Buffer A consisted of 10 mM ammonium formate and buffer B consisted of 10 mM ammonium formate with 90% acetonitrile; both buffers were adjusted to pH 10 with ammonium hydroxide as described previously 44 (link). A CBS-B programed multifunction automatic fraction-collecting instrument (Huxi instrument, Shanghai, China) was coupled to the HPLC and used to collect eluted peptides. The total number of fractions collected was 36 and concatenated to 19 (pooling equal interval RPLC fractions). Ammonium hydroxide and/or ammonium formate were evaporated in a SpeedVac.
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Fractionation of TMT-labeled Peptides
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Corresponding Organization : Chinese Academy of Sciences
Other organizations : Center for Excellence in Molecular Cell Science, Shanghai Institutes for Biological Sciences
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Variable analysis
independent variables
- Fraction collection method (Coupled CBS-B programed multifunction automatic fraction-collecting instrument)
- Number of fractions collected (36 total, concatenated to 19)
- HPLC column (Waters XBridge BEH130 C18 3.5 μm 2.1 × 150 mm column)
- HPLC flow rate (0.2 mL/min)
- HPLC buffers (Buffer A: 10 mM ammonium formate, Buffer B: 10 mM ammonium formate with 90% acetonitrile, both adjusted to pH 10 with ammonium hydroxide)
- Sample (Half of the TMT-labeled peptides mixture)
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