Protein Protocols Protein Protocols

Reverse-Phase HPLC Separation 535
3. Methods
3.1. HPLC Separation of Peptides
The TFA acetonitrile buffer system described in Materials, item 3 is an almost
universal reverse-phase solvent system owing to its low-UV absorbance, high resolution,
and excellent peptide solubilizing properties. The gradient we generally use is:
Time % B
0–60 min 2–37.5%
60–90 min 37.5–75%
90–105 min 75–98%
In the case of extremely complex digests (i.e., tryptic digests of proteins that are
above about 100 kDa), the gradient times may be doubled. In general, we recommend
using the lowest flowrates consistent with near-optimum resolution for the column
diameter that is being used (3,4). Hence, we recommend a flowrate of 75 µL/min for
2.0–2.1 mm columns and a flowrate of 0.5 mL/min for 3.9–4.6 mm columns. Immediately
following their collection, all fractions are tightly capped (to prevent evaporation
of the acetonitrile—see Note 6) and are then stored in plastic boxes (USA/Scientific
Plastics, Part #2350-5000) at 5°C. With the reduced flowrate of 75 µL/min, the average
peak detected fraction volume is about 50 µL, which is sufficiently small that, if necessary,
the entire fraction can be directly spotted onto support disks used for automated
Edman degradation. To prevent adsorptive peptide losses onto the plastic tubes, fractions
should not be concentrated prior to further analysis and, after spotting the peptide
sample, the empty tube should be rinsed with 50 µL 100% TFA, which is then overlaid
on top of the sample. If