Making nanocolumns and tips (Proteomics)

The successful production of nanocolumns and tips is vital to the success of an in-line multidimensional LC-MS/MS analysis. Making nanocolumns for LC/LC-MS/MS is a two-step process: first, fused-silica microcapillary tubing is heated and pulled to produce a column with a tip diameter of 2-5 |m, then the column is packed with chromatographic packing material. The choice and volume of packing materials can vary depending on the complexity of the sample, the volume of the sample, and the goal of the separation, but the optimal diameter of the column tip is the same for all electrospray applications. Typically, the flow rate required for the direct analysis of proteins with femtomole scale sensitivity is 200-300 nL min-1. To achieve this, nanocolumns should have a 50-100 um ID, with a tip ID of approx. 5 um. Columns with smaller IDs can clog easily; those with larger IDs result in less-efficient electrospray.

Nanocolumns may be reproducibly pulled using a commercial CO2 laser puller (Sutter Instruments, Novato, CA), but prepulled columns can also be purchased (i.e., New Objective, Woburn, MA). By pulling the capillary at the midpoint of its length, two nanocolumns are produced from one 50-54 cm length of capillary tubing. Before placing the capillary in the puller, the polyimide coating must be burned off a 1-in. center section using an alcohol burner. Bunsen burners should not be used since they burn hotter and can seal the capillary. If the column is not properly pulled, packing material will not load smoothly and chromatography will be poor. The parameters for pulling optimal tips vary from instrument to instrument and need to be determined experimentally. Some typical values are listed in Table 1. A detailed discussion of these parameters and their role in capillary construction is given in the Sutter Instrument P-2000 manual.

Packing materials are loaded onto the nanocolumns through the use of a stainless steel pressurization device or “bomb” (The Scripps Research Institute, and Cytopea, Inc., see Figure 1). This bomb is attached to a helium tank with a high-pressure line and valve. The bomb has a removable lid and the lid and the base each has a groove that holds a viton O-ring to ensure a high-pressure seal when the lid is tightened down. The lid is tightened to the base with five bolts. A Swagelock fitting is located in the center of the lid. This fitting holds a Teflon ferrule that allows the capillary to be inserted down into the bomb and into the microfuge tube. The Teflon ferrule can be tightened to hold the capillary in place and provide a high-pressure seal. The packing material is suspended in methanol and is placed in a microfuge tube that stands upright in the center of the bomb.

Table 1 Typical parameters for pulling a nanocolumn from 100 x 365-|m fused-silica capillary tubing

Figure 1 Pressure loading device that can be used to pack nanocolumns and to load samples onto nanocolumns

For analyzing simple mixtures of proteins, a one-dimensional nanocolumn can be used. In this case, 10-15 cm of C18 reversed-phase (RP) packing material is loaded into the column. However, for more complex mixtures, a biphasic column is employed in which a 7-cm section of RP material is flanked upstream by 3 cm of strong cation exchange (SCX) resin. This configuration allows for multidimensional separation of peptides because peptides are loaded onto and bound to the SCX resin and sequentially “stepped” off with salt pulses, followed by reverse-phase separation of each subset of peptides. Many samples contain competing salts that interfere with the interaction of the peptides with the SCX resin. In these cases, off-line desalting can be performed using a solid-phase extraction column. Alternatively, peptides can be desalted online using a second phase of reverse-phase packing material directly upstream from the SCX. In this method, peptides can be loaded directly onto the column, desalted in the first cycle of the analysis and subjected to multidimensional separation in the subsequent steps. Comparison of single-dimensional, two-phase, and three-phase LC-MS/MS (McDonald et al., 2002) has shown a dramatic increase in the number of peptides identified in the multidimensional runs versus the single dimensional. A larger but less dramatic increase in the number of peptides identified is seen in a three-phase run versus a two-phase. In particular, more hydrophilic peptides are observed in a three-phase column.

Preparation of the nanocolumn:

1. Cut a 50-54 cm length of 100 x 365-|m fused-silica capillary tubing. Hold the center of the tubing over an alcohol burner and burn the polyimide coating off a 5-10 cm section. Remove the charred material by cleaning with a Kimwipe soaked in methanol. The tubing should be clear in this section.

2. Place the length of tubing in the P-200 laser puller so that the clear section is in the mirrored chamber of the puller. In this position, the laser is focused on the center of the tubing and the fused silica can be melted.

3. Select the program on the laser puller that will result in a 3-5 |m ID tip. The heating program should be repeated three times before the tubing is pulled.

Packing the column:

1. Place a small amount (5mg) of Ci8 RP packing material (5-|m Polaris Ci8-A or similar) in a microcentrifuge tube and add 1 mL of methanol. Agitate the tube to create a slurry of the packing material.

2. Place the open microcentrifuge tube into the stainless steel bomb and secure the lid by tightening the five screws.

3. Insert the flat end of the pulled capillary column through the ferrule until it reaches the bottom of the microcentrifuge tube. Tighten the ferrule until the capillary does not move when gently tugged.

4. Adjust the pressure on the helium tank to 400-800 psi. Slowly pressurize the bomb by opening the valve on the high-pressure line. If the bomb is pressurized too rapidly, the microcentrifuge tube can rupture.

5. When the pressure is applied to the bomb, packing material should begin to rise and fill the capillary. If packing material stops filling the capillary before 7-10 cm have been packed, release the pressure on the bomb, loosen the ferrule holding the capillary and gently tap the capillary on the bottom of the microcentrifuge tube. Re-tighten the ferrule and repressurize the bomb. Continue packing until the desired amount of packing material is loaded.

6. If a two- or three-phase column is to be made, release the pressure on the bomb and remove the capillary column. Open the bomb and replace the C18 packing material with a microcentrifuge tube containing a methanol slurry of SCX (5-|m Partisphere strong cation exchanger, Whatman) packing material.

7. Repressurize the bomb and load 3 cm of SCX packing material. For a three-phase column, repeat the procedure, but load 3 cm of C18 packing material.