Protein Identification

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PROTOCOL

Protein Identification by In-Gel Digestion and Mass Spectrometric Analysis Alastair Aitken* and Michele Learmonth Abstract This protocol details a method for the identification of proteins that have been separated by gel electrophoresis. In-gel digestion of the protein bands with trypsin followed by quadrupole ion-trap or other triple quadrupole mass spectrometry techniques is described. The proteins can be identified by database searching of the mass fingerprint of the intact peptides and of the characteristic fragment masses produced by tandem mass spectrometry. Index Entries: Mass spectrometry; ion-trap; gel electrophoresis; proteomics; databases; protein identification.

1. Introduction This chapter describes the analysis of proteins that have been separated by one- (or two-) dimensional gel electrophoresis. In-gel digestion of the protein bands or spots from 1D gels is detailed. Normally the proteins are digested with trypsin which results in basic charges at both the amino terminal and carboxy-terminal end of most peptides due to the specificity of this protease. The N-terminus of the intact protein is commonly blocked by an acetyl or many other modifications and the C-terminus may well not be a lysine or arginine. Doubly-charged peptide species thus predominate. These are of high energy and fragment more easily in tandem (including ion-trap) mass spectrometry (1). This results in substantial sequence information leading to more powerful database analysis. There are programs available such as “Sequest” (2) that allow the databases to be searched directly with the peptide fragment data (tandem MS-MS data). This results in a large saving of time and effort since the data need not be interpreted into tentative peptide sequences, which can be extremely tedious. Glu-C may also

be used in place of trypsin although fewer doublycharged species will result and analysis may be limited to the mass fingerprint data. This is perhaps more suitable to mass analysis on the highly sensitive MALDI-Tof mass spectrometers where singly charged ions predominate and little sequence or mass fragment data are obtained in any case (3).

2. Materials 1. SDS PAGE electrophoresis apparatus. 2. 0.1% colloidal Coomassie Blue, GELCODE, from Pierce Warriner. 3. 0.2 M NH4HCO3. 4. 50% aqueous acetonitrile. 5. Microcentrifuge. 6. Dithiothreitol (DTT). 7. Centrifugal evaporator (“Savant” or “Gyrovap”). 8. Trypsin (Promega sequencing grade, this has been modified by reductive methylation to remove pseudotrypsin activity and is TPCK treated). 9. 25 µL Hamilton syringe. 10. Formic acid. 11. Coated nanospray needle PicoTip from New Objective (Econo 12).

*Author to whom all correspondence and reprint requests should be addressed: Prof. A. Aitken, Department of Biomedical and Clinical Laboratory Sciences, University of Edinburgh, Hugh Robson Bldg., George Square, Edinburgh EH8 9XD. E-mail: [email protected] OR [email protected] Molecular Biotechnology 2002 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2002/20:1/95–97/$10.75

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12. C8 reverse phase column (0.8 mm x 2 mm, 300Å), L.C. packings. 13. MALDI-Tof plates.

3. Method

3.1. SDS Acrylamide Gel 1. Run 1-D or 2-D SDS acrylamide gel under conditions suitable for proteins of interest (see Note 1). 2. Wash gel in 200 mL water for 5 min. Repeat three times. 3. Visualize proteins by staining gel in 0.1% colloidal Coomassie Blue (GELCODE, see Note 2) for approx 1 h. 4. Destain in water for 1–2 h (see Note 3).

3.2. In-gel Digestion 1. Excise stained gel band by cutting out center (most concentrated part of band) to minimize the amount of acrylamide. 2. Incubate three times (in approx 200 µL 0.2 M NH4HCO3/50% acetonitrile) for 30 min each at 30°C to remove SDS. 3. Incubate gel band in DTT (20 mM) in 2–300 µL of 0.2 M NH4HCO3/50% aqueous acetonitrile for 1 h at 30°C to reduce the proteins. 4. Wash three times in ~200 µL 0.2 M NH4HCO3/ 50% acetonitrile. 5. Alkylate cysteine residues (see Note 4) in fresh iodoacetamide (50 mM, see Chapter 51) in ~100 µL, 0.2 M NH4HCO3/50% acetonitrile for 20 min at room temperature in the dark. 6. Wash three times in ~500 µL 20 mM NH4HCO3/50% acetonitrile. 7. Cut band into 1 × 2mm pieces. 8. Centrifuge for 2 min at 10,000 g (or top speed) in microcentrifuge. 9. Cover pieces with acetonitrile (THEY MUST TURN WHITE). 10. Dry gel band completely by centrifugal lyophilization in centrifugal evaporator (~30 min). 11. Rehydrate gel band with trypsin solution (~0.5– 1.0 µg trypsin freshly made up in 60 µL 50 mM NH4HCO3, for 15-30 min at 4°C (see Note 5). 12. Add sufficient digestion buffer (0.2 M NH4HCO3 or 50 mM Tris pH 7.5) to make up to ~100 µL, i.e., enough to cover the gel pieces. 13. Incubate at 32°C, overnight, ~16 h.

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3.3. Peptide Extraction 1. The following day, centrifuge for 2 min at 10,000 g (or top speed) in microcentrifuge. 2. Collect digest buffer from above the gel pieces. 3. Add 100–200 µL 50% acetonitrile to the gel pieces, sonicate ~30min (35–40°C), leave 1 h, centrifuge as before and collect supernatant. 4. Dry the acetonitrile extract to ~20–50 µL in the centrifugal evaporator then combine with the aqueous extracts. 5. This gives a total volume of ~100–150 µL. Store at –20°C. 6. Peptides should be desalted as described below. For mass fingerprinting and/or sequencing by tandem ESMS or MALDI TOF MS modified procedures should be followed (1,3). For nanospray on an ion-trap mass spectrometer, use the protocol described in Subheading 3.4.

3.4. Peptide Identification by Nanospray MS Analysis of Smaller Proteins (less than 60kDa). 1. For nanospray MS, fit the C8 reverse phase column (0.8 mm × 2 mm) with PTFE tubing (3.5 cm) at the inlet end to allow syringe needle to fit snugly. Fit the outlet end with 5.5 cm of 50 µ fused silica tubing (see Note 6) to allow this to insert into nanospray needle. 2. Wash the C8 reverse phase column (0.8 mm × 2 mm) with 200 µL formic acid (0.01% in 95% acetonitrile). 3. Equilibrate the column with ~200 µL aqueous formic acid (0.01%). 4. The sample (dissolved in ~20 µL aqueous formic acid, 0.01%) is loaded slowly onto the column using a 25 µL Hamilton syringe. If the solution is more dilute (i.e., if more sample is required to give good spectra), load up to 3 × 25 µL. 5. Wash column with 15 µL aqueous formic acid (0.01%). 6. Elute directly into the nanospray needle (see Note 7) with aqueous formic acid (0.01%) containing 60% methanol to allow 2 µL to enter needle. 7. After use, store column in 95% aqueous acetonitrile.

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Protein Identification 3.5. Nanospray MS Analysis of Larger Proteins (more than 60kDa). 1. Follow steps 1–5 in Subheading 3.4. 2. Elute directly into the nanospray needle with 2 µL aqueous formic acid (0.01%) containing 20% methanol then in 10% incremental steps of methanol to allow 2 µL to enter needle at each step (see Note 8). 3. If protein is large (i.e., >200kDa) start with aqueous formic acid (0.01%) containing 10% methanol then elute in 5% incremental steps of methanol.

4. Notes 1. SDS-PAGE minigels with 0.5 mm spacers are excellent, because this minimizes the amount of acrylamide in the gel piece. 2. The methanol/acetic acid used in conventional Coomassie blue staining procedures will fix the protein in the gel to a varying degree resulting in much lower recovery. 3. Gel pieces may be stored in water over a weekend. If longer storage required, keep at –20°C. 4. The advantage of alkylating cysteine residues in gel is to avoid difficulty in removing DTT and iodoacetamide when carrying out the reaction in solution. Some researchers alkylate the cysteines with iodoacetic acid which is equally effective and is a matter of personal choice.

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97 5. The solution is initially kept at 4°C until the gel is swollen to minimize autodigestion of trypsin. A rough guide is to use an enzyme substrate ratio of about 1:10. Use 1–3 µL (0.4 to 1.2 µg) trypsin solution from stock made by adding 50 µL NH4HCO3 (0.2 M) to one vial (20 µg). Do NOT use the Promega resuspension buffer supplied (50 mM acetic acid). In view of the inadvisability of storing the trypsin solution at this high dilution, better results are obtained if a large number of samples are processed simultaneously. 6. Use 1/32" ferrules with a reducing ferrule fitting for the fused silica. 7. Preopened needles (PicoTip) from New Objective offer many advantages. There is no need to open by pressing against the inlet. Otherwise there is a risk of loss of precious sample if the tip breaks badly. 8. This reduces the otherwise large number of peptides in each fraction.

References 1. Jensen, O,N., Wilm, M., Shevchenko, A., and Mann, M. (1999) Peptide sequencing of 2-DE gel-isolated proteins by nanoelectrospray tandem mass spectrometry. Methods Mol. Biol. 112, 571–588. 2. Yates, J. R. 3rd (1998) Database searching using mass spectrometry data. Electrophoresis, 19, 893–900. 3. Costello, C. E. (1999) Bioanalytic applications of mass spectrometry. Curr. Opin. Biotechnol. 10, 22–28.

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