Protocol

Analysis of Complex Protein Mixtures Using Multidimensional Protein Identification Technology (MuDPIT)

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

This protocol describes the analysis of a proteome using multidimensional protein identification technology (MuDPIT), which couples 2D-LC to MS/MS, to resolve and identify peptides from complex mixtures. In this method, a pulled capillary microcolumn is packed with two independent chromatography phases: a strong cation exchanger and reversed-phase matrix material. Once a complex peptide mixture is loaded onto the system, no additional sample handling is required, because, as the peptides elute from the column, they are directed into the ESI ion-trap mass spectrometer, where they are ionized, mass-selected, and fragmented. Finally, advanced search algorithms match the fragmented peptides to their respective proteins in a database.

MATERIALS

Reagents

Acetonitrile/acetic acid solution

Ammonium bicarbonate (1 M and 100 mM), freshly prepared

CaCl₂

Dithiothreitol (DTT)

Endoproteinase Lys-C (sequencing grade)

Methanol (HPLC grade)

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

Iodoacetamide (IAA)

Modified trypsin (sequencing grade) (optional, see Step 7)

Poroszyme-immobilized trypsin (Applied Biosystems) (Optional, see Step 7)

Soluble protein fractions

If proteins found in insoluble fractions are to be analyzed using MuDPIT, begin with the steps outlined in Digestion of Insoluble Protein Fractions for MuDPIT analysis.

Chromatography solvent A

Urea

Equipment

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.

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

Fused-silica capillary (size and length depend on the desired split flow rate, see Step 10)

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

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

Movable Plexiglas stage containing Nano-LC ion sources (ThermoFinnigan, Scripps Research Institute, Cytopea, Inc.) (see Fig. 2)

PEEK MicroCross, Microtight tubing sleeves (Upchurch Scientific)

Quaternary HPLC pump (e.g., a quaternary Hewlett-Packard 1100 series)

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

Strong cation exchange resin (e.g., PartiSphere SCX, Whatman)

Tandem mass spectrometer

A variety of mass spectrometers are suitable, including the LCQ Classic, Deca, Duo, or TSQ series (ThermoFinnigan), or the QTOF1 or QTOF2 (Micromass, Inc.).

ThermoFinnigan LCQ Xcalibur software

Water bath preset to 50°C

Incubator, with a rotating wheel set up, preset to 37°C

METHOD

Digestion of Soluble Protein Extracts for MuDPIT

1. Adjust the pH of the protein extract to 8.5 with 1 M ammonium bicarbonate. Determine the protein concentration.
The concentration of the protein mixture is needed for future reference and to determine the amount of protease to add in Steps 5 and 7.

2. Add solid urea to the protein solution to a final concentration of 8 M.

3. Add DTT to the protein solution to a final concentration of 1 mM. Incubate the solution for 20 minutes at 50°C.

4. Allow the solution to cool to room temperature. Add iodoacetamide to the denatured protein to a concentration of 10 mM. Incubate the solution for 20 minutes at room temperature in the dark.

5. Add endoproteinase Lys-C to a final ratio of substrate to enzyme of 100:1 and incubate the reaction overnight at 37°C.

6. Dilute the sample to 2 M urea with 100 mM ammonium bicarbonate (pH 8.5). Add CaCl₂ to a final concentration of 1 mM.

7. Digest the protein mixture with trypsin using either of the following two methods.

For Method A:

i. Add modified trypsin to the protein solution to a final ratio of substrate to enzyme of 50:1. Incubate the reaction overnight at 37°C.

ii. Centrifuge mixture in a microfuge at maximum speed to remove insoluble material.

iii. Transfer the supernatant to a fresh microfuge tube.

For Method B:

i. Add ~1 µl of Porosyzme immobilized trypsin slurry to every 50 µg of protein starting material. Incubate the reaction overnight at 37°C on a rotating wheel.

ii. Centrifuge the reaction in a microfuge at maximum speed to remove insoluble material and the trypsin beads.

iii. Transfer the supernatant to a fresh microfuge tube.
Method B has the advantage that there is less trypsin contaminating the protein sample during the subsequent analysis.

8. Load the supernatant onto a SPEC Plus PT C₁₈ solid-phase extraction cartridge according to the manufacturer’s instructions. Exchange the supernatant into acetonitrile/acetic acid solution (5% acetonitrile/0.5% acetic acid).
This essential step removes buffer components from the complex peptide mixture (i.e., ammonium bicarbonate, urea, etc.) that are incompatible with ESI-MS/MS. Removal of salt from the sample is critical, because salt prevents the peptides from binding to the strong cation-exchange portion of the 2D column. A sample containing 1 M salt will fail in a MuDPIT analysis. In addition, the extraction cartridge concentrates the sample, which is important because it can take ~1.5 hours to load 15 µl onto the 2D microcolumn.

MuDPIT

To carry out MuDPIT, a system must be assembled with a tandem mass spectrometer and a quaternary HPLC pump. This protocol has been run using a quaternary Agilent HP1100 series HPLC, directly coupled to a ThermoFinnigan LCQ Deca ion-trap mass spectrometer equipped with a nano-LC-ESI source.

9. Prepare the column as described in Analysis of Complex Protein Mixtures Using Nano-LC Coupled to MS/MS. In summary:

i. Pull a fused-silica capillary microcolumn (100 µm I.D. x 365 µm O.D.) with a P-2000 laser puller.

ii. Pack the microcolumn with 10 cm of C₁₈ reversed-phase material in methanol.

iii. Pack the column with 4 cm of 5 µm strong cation-exchange material in methanol.

10. Connect the column to an Upchurch PEEK MicroCross (for details, see Analysis of Complex Protein Mixtures Using Nano-LC Coupled to MS/MS, Step 4, and Fig. 1 ) to which is also attached a gold wire to supply a spray voltage of ~1800 V, a microcapillary split line to provide an effective flow rate of 100-300 nl/minute, and a solvent line from the HPLC.

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Figure 1. 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.

Measure the flow from the tip of the microcolumn by following Analysis of Complex Protein Mixtures Using Nano-LC Coupled to MS/MS, Step 6.

11. Equilibrate the column with 100% solvent A for 5 minutes at a flow rate of 150 µl/minute at the pump.

12. Follow Analysis of Complex Protein Mixtures Using Nano-LC Coupled to MS/MS, Steps 12-17 to load the sample onto the column.
Because MuDPIT columns are longer than single-phase LC microcolumns, a pressure of ~1000 psi (provided by a helium tank with regulator) is required to load the sample.

13. Place the MicroCross with the connections into a stage, as per Analysis of Complex Protein Mixtures Using Nano-LC Coupled to MS/MS, Steps 18 and 19, and Movable Plexiglas stage containing nano-LC electrospray power source (see Fig. 2 ).

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Figure 2. Movable Plexiglas stage containing nano-LC electrospray power 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 up 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 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 spectropmeter, allowing electrospray ionozation to occur. An XYZ manipulator is used to provide fine positioning of the microcolumn with respect to the entrance of the mass spectrometer.

14. Begin the MuDPIT analysis of the peptide sample.
The key feature of MuDPIT is the 2D capillary microcolumn chromatography, which performs better than when the resins are in separate columns and the buffers shunted through the columns. By having the beds integrated into the same column, the sensitivity is much higher and the sample losses are much fewer. The chromatography is controlled by the mass spectrometer itself, through the use of Xcalibur software. A typical analysis is a fully automated, 15-step chromatography run on highly complex mixtures, but any number of steps can be applied to a column. The gradient of each individual step is built within the Instrument Setup portion of Xcalibur (refer to Analysis of Complex Protein Mixtures Using Nano-LC Coupled to MS/MS, Steps 20 and 21, and Tandem Mass Spectrometry Analysis Using the ThermoFinnigan LCQ System). A typical 15-step MuDPIT setup is shown in Table 1.
Typically, the instrument is operated here in a very similar manner to that described in Steps 20 and 21 in Analysis of Complex Protein Mixtures Using Nano-LC Coupled to MS/MS; and Tandem Mass Spectrometry Analysis Using the ThermoFinnigan LCQ System; however, instead of the top two ions being selected for MS/MS, the top three ions are selected during MuDPIT analysis.