Target Discovery and Validation Reviews and Protocols

Pancreatic Cancer 81
in many ways. However, two different strategies are frequently used for separation
of peptides. One setup uses two columns in tandem (a precolumn followed
by an analytical column), whereas the second setup only uses a single analytical
column. The tandem column setup is preferred when large volumes (10–40 µL)
have to be analyzed or if the samples need to be desalted or washed extensively.
This setup is very robust and can be used for most types of samples. One
drawback is that one might loose sensitivity because of broader chromatographic
peaks. Several column materials can be used for precolumns (e.g.,
YMC ODS-A, 5- to 15-µm beads; Kanamatzu USA Inc., New York, NY) and
analytical columns (e.g., MS218, 5-µm beads; Vydac) and it is therefore
strongly suggested that several trial runs including different reverse phase
materials are tested for obtaining optimal separation and resolution during the
LC-MS/MS analysis.
The following sections describe a general strategy for building and packing
reverse phase columns used for nano-LC-MS/MS analysis in addition to some
general chromatographic guidelines (LC program, length of gradient, and
context of mobile phase).
3.3.9. Preparation of Reverse Phase Column (Fig. 2)
1. The column is packed in a fused silica capillary tubing (375 µm od and 75 µm id)
(52). A “frit” restrictor has to be generated inside the fused silica to block the
reverse phase material during packing (the silica tubing is open in both ends). Mix
44 µL of Kvasil 1 with 8 µL of formamide and vortex for 1–3 min (the solution
becomes viscous).
2. One end of a 20- to 25-cm fused silica is dipped in the solution for 1 to 2 s (the
solution will move upward into the fused silica by capillary action). Excess solution
is wiped off and the fused silica is left at room temperature o.n. to polymerize.
The frit is checked under a microscope and should be approx 2–5 mm in
lengths and has to present a V-shaped cone pointing outward. The frit can be cut
if it is too long.
3. Insert the fused silica into a pressure vessel and wash it with 100% methanol. This
process ensures that solvent can flow through the frit and that when pressure is
applied, the frit is still affixed to the capillary. The reverse phase material (~4 mg)
is placed in a glass microvial and resuspended in 500 µL of methanol.
4. To keep the column material in suspension a small magnetic stirring rod is added
to the glass microvial before placing it in the pressure vessel. Place the pressure
vessel on a stir plate and turn the magnetic stirrer at low speed. Turn the gas (He)
pressure to 50 bar (800 psi), which initiates the packing of the column. The column
should be 5–10 cm for precolumns and 15–20 cm for analytical columns.
5. The fused silica is connected to the LC system and flushed with mobile phase B
(90% acetonitrile, 0.4% acetic acid, and 0.005% heptafluorobutyric acid in water)
for 30 min and subsequently 30 min with mobile phase A (H2O with 0.4% acetic
acid and 0.005% heptafluorobutyric [v/v]) to equilibrate the column.