Protocol

Analysis of Complex Protein Mixtures Using Nano-LC Coupled to MS/MS

This protocol was adapted from "The Use of Mass Spectrometry in Proteomics," Chapter 8 in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

Nanoliter-LC coupled to tandem mass spectrometry (nano-LC-MS/MS) permits the rapid and sensitive determination of protein-protein interactions. By using a specific purification technique such as coimmunoprecipitation or affinity purification in conjunction with nano-LC-MS/MS, not only are proteins identified, but specific protein-protein interactions are elucidated as well.

MATERIALS

Reagents

Acetic acid

Acetonitrile (HPLC grade)

Acetonitrile/formic acid solution

Helium gas (supplied by tank with regulator; at least 1000 psi pressure)

Methanol (HPLC grade)

Protein fractions to be analyzed

Chromatography solvent A

Chromatography solvent B

Equipment

Alcohol burner

C₁₈ reversed-phase packing material (5 µm) (e.g., Zorbax XDB, Agilent Technologies)

CAUTION: Do not inhale; use in a chemical fume hood.

C₁₈ solid-phase extraction pipette tips (e.g., SPEC Plus PT C18, ANSYS Technologies)

These C₁₈ solid-phase disk pipette tips have a 0.4 µg sorbent capacity and a loading volume of up to 800 µl.

Ceramic scribe

Fused-silica capillary (100 µm I.D. x 365 µm O.D.) (Agilent Technologies or Polymicro Technologies)

Fused-silica capillary (50 x 365 µm)

Fused-silica capillary of a suitable size and length (see Step 4.ii)

Gold wire (0.025 diameter) (Scientific Instrument Services, Inc.)

Graduated glass capillaries

High-performance liquid chromatography (HPLC) equipment

Suitable software for setting up HPLC gradient profile (such as the ThermoFinnigan LCQ Xcalibur software, see Step 20)

Laser puller (e.g., P-2000, Sutter Instruments)

Movable Plexiglas stage containing Nano-LC ion sources (ThermoFinnigan, Scripps Research Institute, or Cytopea, Inc.) (see Step 18 and Fig. 6)

PEEK MicroCross, Microtight tubing sleeves (Upchurch Scientific)

Stainless steel pressurization bomb (Scripps Research Institute or Cytopea, Inc.)

Tandem mass spectrometer (e.g., ThermoFinnigan, Micromass)

METHOD

Preparation of a Nano-LC Column

1. Make a window in the center of an ~12-15-inch, 100 x 365-µm fused-silica capillary by holding it over an alcohol flame until the polyimide coating has been charred (see Fig. 1 ). Remove the charred material by wiping the capillary with a tissue soaked in methanol (see Fig. 2 ).

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Figure 1. Preparation of a window in the fused-silica capillary. The capillary is held over an alcohol flame to char the polyimide coating. The length of the charred portion is ~1-3 cm.

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Figure 2. Charred portion of the coating on the capillary is removed by wiping it away with a tissue soaked in methanol. All of the burned polyimide coating must be removed to prevent deposition on the laser puller’s retro-mirror when the laser is focused on the newly made window.

Unlike an alcohol burner, a Bunsen burner flame is too hot and will seal the inside of the capillary.

2. To pull a needle, place the window portion of the capillary into the P-2000 laser puller (see Fig. 3 ). Position the window in the mirrored chamber of the puller, where the laser will concavely focus and melt the fused silica.

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Figure 3. The fused-silica capillary is placed into the laser puller to produce two pulled-needle capillaries. The windowed area of the capillary is placed within the "shroud" containing the retro-mirror. The ends of the capillary are fastened within the vises to hold the capillary in position.

Arms on each side of the mirror have grooves and small vises, which properly align the fused silica and hold it in place. See Table 1 for advice on suitable parameters.

3. Pack the pulled-needle capillary with C₁₈ reversed-phase packing material using a stainless steel pressurization bomb.
IMPORTANT: Always wear safety glasses during this step. In the event that the capillary has not been seated properly, it will be forced out of the bomb at great velocity due to the pressure.

i. Place ~5 mg of C₁₈ reversed-phase packing material into a 1.7-ml microfuge tube and add ~1 ml of methanol. Shake the tube to suspend the particles and place it into the bomb. Secure the bomb lid by tightening the five bolts.
It is very important to secure all five bolts. Both the lid of the bomb and the bomb itself have grooves that fit an O-ring; this provides an air-tight seal when the lid is seated against the bomb.
WARNING: High-pressure gas will escape violently if the lid is not secured tightly.

ii. The lid has a Swagelok fitting containing a Teflon ferrule. Feed the fused-silica, pulled-needle capillary down through the ferrule until the end of the capillary reaches the bottom of the microfuge tube. Tighten the ferrule to secure the pulled-needle capillary (Fig. 4 shows the pressurization bomb and the microcapillary to be packed with reversed-phase material).

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Figure 4. Pressurization device or "bomb" with high-pressure line and valve. The inner portion of the bomb contains an open area to allow a microcentrifuge tube to stand upright with the cap open. The bomb and the lid have a groove, which holds a viton O-ring to ensure a high-pressure seal when the lid is tightened down. The lid contains five holes for bolts and, in the center, a Swagelok fitting. Within the fitting sits a Teflon ferrule that allows the capillary to be inserted down into the bomb and into the microcentrifuge tube. The ferrule is tightened to hold the capillary in place and provides a high-pressure seal.

iii. Apply pressure to the bomb by first setting the regulator on the helium gas cylinder to ~400-800 psi and then opening a valve on the bomb to pressurize it.
The packing material will begin filling the pulled needle capillary. This now becomes the capillary microcolumn. To achieve good chromatographic separation, pack the capillary with 10-15 cm of material.

4. Attach the needle to the HPLC system through an Upchurch PEEK MicroCross. For a layout of the connections for the Upchurch MicroCross, see Figure 5 .

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Figure 5. Layout of the Upchurch MicroCross. The first connection at the bottom is for a transfer line to bring the solvent flow from the HPLC pump to the MicroCross. Moving clockwise to the second connection is the split line, which is used to control the final flow rate of the solvent through the microcolumn. The next connection is to hold a small section of gold wire, which makes electrical contact with the solvent. The final connection is where the microcolumn is attached.

i. The first connection point of the cross contains the transfer line from the HPLC pump. This consists of a 50 x 365-µm fused-silica capillary, where the length is sufficient to reach from the HPLC pump to the mass spectrometer.

ii. The second connection point contains a length of fused-silica capillary that is used as a split line. This split line allows a majority of the flow to exit through the split; therefore, very low flow rates can be achieved through the packed capillary microcolumn. The size and length of this section of capillary depend on the flow rate from the pump and the length of the microcolumn. A good starting point is to use a 2-foot section of 100 x 365 µm for the split line (but see Step 6).

iii. The third connection point contains a section of gold wire, which will raise the voltage of the solvent entering the needle from zero to ~1800 V, thus allowing electrospray to occur.

iv. The fourth connection point is for the packed capillary microcolumn.

5. Before loading the sample, equilibrate the column by pushing the methanol out with 100% solvent A for 5 minutes at a flow rate of 150 µl/minute at the pump.

6. After 5 minutes, measure the flow from the tip of the capillary microcolumn, using graduated glass capillaries. The target flow rate at the tip should be ~100-300 nl/minute. If the flow rate is above this value, use a ceramic scribe to cut off a portion of the split line capillary. This will allow more of the flow to exit out of the split and less flow through the microcolumn. If the flow is less than 100 nl/minute, use a longer piece of capillary or a section with a smaller inner diameter to force more flow through the microcolumn.
Measuring the flow rate and adjusting the split line may have to be repeated a number of times until the target flow rate is reached.

Concentration of Sample

Peptide samples can be solubilized in any number of reagents, including Tris, ammonium bicarbonate, acetic acid, formic acid, and urea. However, peptide samples are typically the product of a digested protein or protein mixture, in which a variety of reagents may be present including some that will interfere with the performance of the reversed-phase column and the mass spectrometer (see the note below Step 11).

Peptide sample volumes range from several microliters to 1 ml or more. The bed volume of the microcolumn is ~1.5 µl, which allows samples up to 50 µl to be loaded directly onto the column. For sample volumes greater than 50 µl, concentration of the sample (as detailed in Steps 7-11) is necessary.

7. Wet the SPEC Plus PT C₁₈ solid-phase extraction pipette tip with 1 ml of methanol as follows:

i. Push approximately half of the methanol through the disk, and then wait for 15-30 seconds to allow the disk to activate.

ii. Push the remainder of the methanol through, but do not push air through the disk.

8. Equilibrate the disk with 1 ml of solvent A by pushing it through the disk.

9. Pull the peptide solution into the pipette tip and then push the solution back out.
This can be repeated two to three times. Peptides remain in the tip and are concentrated onto the disk. The flowthrough can be discarded.

10. Elute the peptides with ~100 µl of 90% acetonitrile/0.5% acetic acid. Push the eluting solvent through the disk, pull the solution back up through the disk, and then push it through one final time, to give three passes across the disk.

11. Use a vacuum concentrator to remove the acetonitrile until the peptides are nearly dry. Resuspend the peptides in ~10-15 µl of acetonitrile/formic acid solution. The sample is ready to load onto the reversed-phase capillary microcolumn.
Avoid detergents whenever possible or remove them prior to loading the sample onto the reversed-phase column. Once a detergent enters the column, it will bind to the reversed-phase and "leak off" in the elution gradients, contaminating subsequent analysis. Detergents ionize more readily than peptides and therefore will mask any peptide ions.
(see Troubleshooting)

Loading the Sample onto the C18 Column

12. Centrifuge the samples in a microfuge at 14,000 rpm for 10 minutes to pellet any solid material.

13. If any pellet is visible, transfer the supernatant (peptide sample) to a fresh microfuge tube. Even minute amounts of solid material will plug the microcolumn. Place the tube containing the peptide sample into the pressurization bomb, and tighten the lid to the bomb (for instructions on tightening the bomb lid, see Step 3).

14. Feed the reversed-phase capillary microcolumn down through a Teflon ferrule until the end of the capillary reaches the bottom of the microfuge tube. Tighten the ferrule to secure the capillary microcolumn.

15. Set the regulator on the gas cylinder to 400-800 psi, and then pressurize the bomb by opening the valve on the bomb that connects to the gas cylinder. The peptide sample will begin to flow into the column.

16. Measure the loaded volume at the tip of the needle using a graduated glass capillary.

17. When 8-10 µl are loaded, release the pressure, remove the column from the bomb, and place it back into the Upchurch MicroCross.

Ion-Source Setup

18. Place the Upchurch MicroCross with the connections into a stage, which is designed in this case for the ThermoFinnigan LCQ series mass spectrometer. This stage performs a threefold purpose:

i. It supports the MicroCross and holds it in place along with the connections.

ii. It insulates the MicroCross from electrical contact with its surroundings when it is held at high-voltage potential.

iii. It allows for fine position adjustment of the microcolumn with respect to the entrance of the mass spectrometer (heated capillary) by using an XYZ manipulator.
Figure 6 shows the stage comprising the Plexiglas support, MicroCross, high-voltage connection, microcolumn, and entrance to the mass spectrometer. Plastic tabs with small Teflon screws are used to hold the connections in place. The potential needed to raise the solvent in the cross and the microcolumn to 1800 V is delivered through an insulated cable that attaches to an aluminum block seated in the Plexiglas support. Electrical contact is made when the plastic tab is tightened down onto the gold wire, which presses down onto the aluminum. This allows only the MicroCross to be energized and not the XYZ manipulator or metal support plate.

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Figure 6. Movable Plexiglas stage containing nano-LC electrospray ion source. The Upchurch MicroCross with HPLC connections is held in position in the Plexiglas stage with plastic tabs. The solvent enters the MicroCross from the transfer line of the HPLC pump. A majority of the flow leaves the cross through the split line, but a small fraction moves through the microcolumn toward the opening of the mass spectrometer. An insulated cable supplies the high voltage that is connected to the aluminum portion of the stage. The aluminum makes contact with the gold wire, energizing the solvent flowing through the cross. This provides a large voltage potential between the tip of the microcolumn and the opening of the mass spectrometer, allowing electrospray ionization to occur. An XYZ manipulator is used to provide fine positioning of the microcolumn with respect to the entrance of the mass spectrometer.

19. Position the microcolumn using the XYZ manipulator so that the needle tip is ~2-5 mm from the orifice of the mass spectrometer’s heated capillary, and set the voltage at 1.5-1.8 kV.

HPLC Programming

20. Program the HPLC system as follows:

i. Start the gradient with 100% solvent A (0% solvent B) and a flow rate of 150 µl/minute.

ii. Over a period of time (call it X minutes), ramp up the concentration of solvent B to 60% along with an increase in the flow rate to 250 µl/minute.

iii. Set a ramp-down period of 5 minutes, during which the concentration of solvent B returns to 0% and the flow rate slows to 150 µl/minute.
As a guide, if the sample contains ~15-20 proteins, X should be a 30-minute gradient; if the sample contains ~40-50 proteins, X would be either a 60- or 90-minute gradient. The gradient profile can be programmed through the ThermoFinnigan Xcalibur software or the Micromass Inc. Masslynx software, depending on the mass spectrometer being used.

21. If the ThermoFinnigan LCQ Xcalibur software is being used, use the settings in Tandem Mass Spectrometry Analysis Using the ThermoFinnigan LCQ System to create a typical data-dependent MS/MS method. For experimental design of data-dependent tandem mass spectra acquisition for the QTOF2 Masslynx software, refer to the Masslynx manual (version 3.5).

TROUBLESHOOTING

Problem: Sample is viscous and difficult to load.

[Step 11]

Solution: If the sample contains solubilization or fractionation agents such as urea, glycerol, or sucrose, loading may be somewhat difficult due to the viscosity of the solution. In such cases, once the sample is loaded, wash the column extensively with 100% HPLC solvent A to remove these chemicals before starting the gradient.