RNA Epitranscriptomics and Proteomics Resource

SDS-PAGE separation and digestion of proteins for LC-MS analysis

Sample preparation standard operating procedure (SOP)

Author: Dr. Qishan Lin

Date: 03/30/2018

Summary: This SOP describes the steps necessary to carry out SDS-PAGE separation of proteins and in-gel digestion. The protocol uses MS-compatible gel stain to facilitate subsequent analysis by LC-MS and LC-MS/MS, which is described in a separate SOP.

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Standard workflow - Securing initial sample (for example, by cell lysis, immunoprecipitation, FPLC, anything that may be necessary for the task at hand). - Separation by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE). - Staining with MS-compatible stain. - Band excision. - Extensive de-staining, if no MS-compatible stain was employed. - In-gel reduction and alkylation. - In-gel enzymatic digestion with sequencing-grade trypsin. - Digest concentration.

Safety considerations Gloves and protective clothing should be worn throughout.

Reagents and Equipment a) Reagents: SDS and ammonium bicarbonate (Sigma, St. Luis, MO); acetonitrile,

formic acid, trifluoroacetic acid (TFA) (Sigma, MO); dithiothreitol (DTT), iodoacetamide, tris-(hydroxymethyl) aminomethane (Tris). Brilliant Blue G-250 (any manufacturer stain would be fine); Sequencing grade trypsin (Promega, Madison, WI). All solutions are prepared by using Milli Q water, or HPLC grade water (Sigma or Fluka).

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b) Equipment: Microcentrifuge (with rotor for 1.5 ml tubes); vortexer; water bath or

heating block capable of reaching 100°C; thermomixer (e.g., Eppendorf, Hamburg, Germany). Gel electrophoresis apparatus (e.g., Bio-Rad)

SDS PAGE separation

- Use a typical 12% SDS-PAGE under reducing conditions. - Load a minimum of 30 µg samples of the protein mixture dissolved in SDS-PAGE sample buffer. - Run the separation by using appropriate voltage/current. - Stain the gel with Brilliant Blue G-250.

Band excision, in-gel reduction and alkylation a) Reagents: 5.7 mg Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in 1,000 µL of 100 mM NH4HCO3; 7.5 mg iodoacetamide (IDA, Sigma) in 1000 µL of 100 mM NH4HCO3; 100 mM NH4HCO3.

b) Procedure: 1. Keep gel at 4°C in 1% acetic acid.

2. Excise the spot using a scalpel, or a #18-gauge needle. Don’t forget to cut a "control" piece of gel.

3. Wash the gel piece with 200 µL of deionized H2O.

4. Spin down, discard the supernatant, and add 200 µL of 100mM NH4HCO3.

5. Spin down, discard the supernatant, and add 200 µL of acetonitrile. 6. Vortex, spin for 5 min.

7. Discard the supernatant, and add 200 µL of acetonitrile. 8. Spin down and remove the liquid, repeat. 9. Speed-vac briefly.

10. Add 50 µL of reducing reagent (make sure pH is above 8).

11. Incubate at 37°C for 30 min.

12. Spin gel pieces down and remove excess liquid.

13. Shrink the gel with 200 µL of acetonitrile. Repeat.

14. Add 50 µL of IDA and incubate for 30 min in dark at 37°C.

15. Remove the IDA solution.

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16. Shrink the gel with 200 µL of acetonitrile. Repeat. 17. Spin and remove all the liquid. 18. Shrink the gel as before and submit to speed-vac, lyophilization, or other

method to eliminate solvent. In-gel trypsin digestion Procedure:

1. Swell the gel pieces in a digestion solution consisting of 12.5 ng/µL of trypsin (Sigma, Proteomics grade) in 50 mM NH4HCO3. Keep the solution in an ice-cold bath for 45 min.

Note: the trypsin stock consisted of a 0.5µg/µL solution in 50 mM acetic acid, which was subsequently diluted according to these proportions: 4 µL stock + 76 µL of deionized water + 80 µL of 100 mM NH4HCO3. 2. Remove the extra supernatant.

3. Replace with 5-10 µL of the digestion solution (50 mM NH4HCO3)

4. Keep the gel pieces wet during enzymatic cleavage at 37°C overnight.

5. Extract the peptides by exchange with 15 µL of 20 mM NH4HCO3. 6. Spin, remove the supernatant and transfer to a new siliconized tube (low

retention tube).

7. Extract three times by exchange with 20 µL aliquots of 5% formic acid and 50 % acetonitrile at room temperature for 20 min each. Gentle sonication will help recover the peptide products from the gel.

8. Pool the exchanged fractions (i.e., ~15 + 3×20 µL).

9. Speed-vac or otherwise concentrate to a final volume of 1-2 µL. 10. The sample is now ready for LC-MS and LC-MS/MS analysis.

Important considerations - For best results, each band should contain at least a few nanograms or more of total protein. The amount of protein in the band can be estimated by using Coomassie blue staining and comparing with the gel provided in the below figure. - Faint bands visualized by silver staining do not contain sufficient material to enable identification by LC-MS and LC-MS/MS analysis (see below figure).

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- For best results, bands should be visualized by using a MS-compatible stain (e.g., Brilliant Blue G-250). If other stains are employed, extensive de-staining should be carried out after band excision, but before in-gel digestion.

- A schematic overview of the in-gel digestion procedure followed by LC-MS analysis. Protein sample is separated by 1D SDS-PAGE, and the lane is cut into bands for parallel processing. Reduction and alkylation are performed prior to enzymatic digestion of proteins. Peptides are extracted for LC-MS/MS analysis followed by protein sequence database searches (Methods Mol Biol. 2014; 1156: 53–66)

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REFERENCE “Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels,” A. Shevchenko, M. Wilm, O. Vorm, and M. Mann, Anal. Chem., 1996, 68 (5), 850-858