Protocol

HPLC Separation of Digested Proteins and Preparation for Matrix-Assisted Laser Desorption/Ionization Analysis

This protocol was adapted from "Identification of Novel Protein Complexes and Protein-Protein Interactions by Mass Spectrometry," Chapter 18, in Protein-Protein Interactions , 2nd ed. (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

Two types of columns are commonly used for the separation of peptides by HPLC. A single-phase column contains the reverse-phase resin C18, which interacts with the hydrophobic moieties of the peptides. Peptides resulting from digestion of simple mixtures of proteins are loaded onto the single-phase column and eluted into the mass analyzer using an increasing gradient of an organic solvent. Peptides resulting from the digestion of more complex mixtures of proteins are resolved using a biphasic column. This column integrates both a strong cation exchange SCX resin, which interacts with peptides as a result of their positive charge, and a reverse-phase C18 resin, packed in tandem. Peptides initially interact with the SCX resin and are eluted into the C18 resin by ammonium acetate that competes for the peptide-binding sites. Peptides are then eluted from the C18 resin into the mass analyzer. This process is repeated using increasing concentrations of ammonium acetate to differentially elute peptides in a stepwise fashion. The biphasic column also uses an additional C18 reverse-phase resin linked through an Inline MicroFilter Assembly (Upchurch) to desalt the peptides prior to loading onto the SCX. The desalting and biphasic columns are combined to give an integrated desalting/biphasic column.

RELATED INFORMATION

See Overview of Affinity Purification in Combination with Mass Spectrometry.

Materials

Reagents

Acetonitrile

Aqua 5-µm C18 (Pheromenex)

Dihydroxybenzoic acid (DHB)

Methanol

Milli-Q water

Peptide elution buffer A

Peptide elution buffer B

Peptide elution buffer C

Protein sample, digested (see Direct Enzymatic Digestion of Protein Complexes for MS Analysis)

SCX (strong cation exchange) Partisphere 5 (Whatman)

Equipment

Bunsen burner

Equipment for degassing solutions

Equipment for HPLC

Fused silica capillary tubing, 100-µm inner diameter (i.d.), 365-µm outer diameter (o.d.) (Polymicro Technology)

Fused silica capillary tubing, 250-µm i.d., 365-µm o.d. (Agilent)

Inline MicroFilter Assembly (Upchurch)

Kimwipes

MicroTee Assembly (Upchurch)

MicroTight Green Sleeve (Upchurch)

Sutter Instrument P-2000 CO₂ laser puller (Novato)

Stainless steel plate

Microcolumn Preparation

1. Take 45 cm of 100-µm i.d. capillary tubing. Using a Bunsen flame, burn off 2-3 cm of the polyimide coating in the middle of the capillary tubing.

2. With a methanol-wetted Kimwipe, remove the burnt polyimide to expose the underlying silica.

3. Place the capillary tubing in a Sutter Instrument P-2000 CO₂ laser puller with the exposed silica area in line with the laser. Use the laser puller to produce two columns, each with a length of 22.5 cm and a 5-µm tip.

4. Prepare an Aqua 5-µm C18 slurry by combining a spatula tip of C18 material with 500 µl of methanol. Vortex the slurry.
For single-phase columns:

i. Pack 10-12 cm of Aqua 5-µm C18 into one of the tips obtained in Step 3.

ii. Degas peptide elution buffers A and B.

iii. Equilibrate the column by running a gradient of 60% buffer B to 100% buffer A over a 30-minute period, followed by an additional 15 minutes of 100% buffer A.
Use an applied flow rate of 0.1 ml/min and a tee splitter to reduce the flow rate to 300-400 nl/min.

For integrated biphasic columns:

i. Make a desalting column by inserting 12 cm of 250-µm i.d. capillary tubing into an inline microfilter assembly (closest to the filter). Use two microtight green sleeve adapters to match the diameter of the 250-µm i.d. capillary tubing to the inline microfilter assembly.

ii. Insert 5 cm of 250-µm i.d. capillary tubing to the front end of the desalting column.
This is a waste line that can be reused.

iii. For the desalting column, pack 4-5 cm of Aqua 5-µm C18 packing material (from Step 4) into the 12-cm, 250-µm capillary tubing.

iv. Make an SCX slurry by combining a spatula tip of SCX material with 500 µl of methanol. Vortex the slurry.

v. Into a 5-µm tip (from Step 3), pack 10 cm of Aqua 5-µm C18 (from Step 4) followed by 2-3 cm of SCX, to generate the biphasic column.

vi. Degas peptide elution buffers A and B.

vii. Equilibrate the desalting and biphasic columns separately by running a gradient of 60% buffer B to 100% buffer A over a 30-minute period, followed by an additional 15 minutes of 100% buffer A. Use an applied flow rate of 0.1 ml/min and a tee splitter to reduce the flow rate to 300-400 nl/min.

Separation of Peptides by HPLC

5. Load the digested protein sample of interest onto the column.

For single-phase columns:

i. Load the digested protein sample directly onto the equilibrated C18 5-µm tip.

ii. Wash the protein-loaded column for 5-10 minutes with buffer A.

iii. Elute peptides from the HPLC with the following gradient:

Time (min) % Buffer A % Buffer B 0 100 0 10 100 0 20 85 15 80 55 45 90 0 100 100 0 100 105 100 0 125 100 0

For integrated biphasic columns:

i. Load the digested protein sample onto the equilibrated desalting column.

ii. Desalt the loaded sample by washing for 5-10 minutes with 100% buffer A.
At this point, the sample is loaded onto the C18 resin of the filter column setup.

iii. Take the equilibrated 5-µm tip and cut at the bottom of the SCX to remove the unloaded portion of the column.

iv. Replace the waste line of the desalting column with the 5-µm tip to yield the integrated biphasic column.

v. Degas peptide elution buffer C.

vi. Start elution of peptides from the HPLC with an ammonium-acetate-free step, followed by gradient elution with buffer B:

Time (min) % Buffer A % Buffer B % Buffer C 0 100 0 0 5 100 0 0 60 55 45 0 70 0 100 0 80 0 100 0 90 0 100 0

vii. Continue elution with a series of increasing ammonium-acetate steps (where X = % buffer C), each followed by gradient elution with buffer B:

Time (min) % Buffer A % Buffer B % Buffer C 0 100 0 0 3 100 0 0 3.1 100 - X 0 X 5 100 - X 0 X 5.1 100 0 0 15 85 15 0 60 75 25 0 112 45 55 0 115 0 100 0 125 0 100 0

The number of ammonium-acetate steps depends on the complexity of the sample, where more complex samples require more ammonium-acetate steps. Typically, four salt steps of 10%, 25%, 50%, and 80% of buffer C are sufficient.

viii. Conclude the elution with a salt step of 100% buffer C and gradient elution with buffer B:

Time (min) % Buffer A % Buffer B % Buffer C 0 100 0 0 2 100 0 0 2.1 0 0 100 15 0 0 100 15.1 100 0 0 23 85 15 0 90 70 30 0 140 35 65 0 150 100 0 0

Matrix-Assisted Laser Desorption/Ionization

6. Prepare a saturated solution of DHB in a 50%/50% (v/v) mixture of acetonitrile and water.

7. Spot a stainless steel plate with 1 µl of protein analyte solution (eluted from either single- or biphasic columns in Step 5) and 1 µl of the DHB mixture.

8. Allow the spot to air-dry.

9. Wash the spot with 2 µl of water by aspirating over the protein spot.

10. Analyze by MALDI/TOF.