Sample Preparation

The success of any mass spectrometry experiment depends on proper sample preparation. MS data quality is extremely dependent on sample quality. There are many salts and detergents that are incompatible with MS that must be avoided from the onset or removed prior to MS analysis.

The proteome is very complex and there is no one standard method for preparing protein samples for mass spectrometry analyses. Individual protocols differ depending on sample type, experimental goals, and analytical method.

Workflows that incorporate optimized cellular lysis, subcellular fractionation, protein/peptide depletion or enrichment, and reproducible protein concentration assays all contribute to the accurate identification and quantitation of proteins. In all cases, the quality and reproducibility of sample extraction and preparation significantly impact your research results.

Users are strongly encouraged to consult with core staff regarding proper sample preparation before starting an experiment.

In-gel digestion procedure


Important considerations

  1. Keratin contamination is a major issue with in-gel digestions because it can compromise the detection of less abundant proteins present in the gel. It is imperative that precautions be taken to avoid contamination by human keratin proteins. Keratin is ubiquitous and comes not only directly from skin and clothing but also dusty surfaces and equipment, including gel staining boxes. Ideally, all gel manipulation prior to trypsin digestion should be done in a laminar flow hood.
  2. Wear clean gloves and lab coat and sleeve protectors. If you touch things outside the hood, change your gloves before continuing.
  3. Reduce dust in the work area by rinsing or wiping down surfaces and equipment, e.g., pipetmen, outside and inside of the Speed Vac and centrifuge, tube racks, bottles.
  4. Keep containers of microfuge tubes separate and tips separate and closed.
  5. You can use disposable 14 cm sterile tissue culture dishes for staining. Never use boxes that have been used for western blotting as some blocking agents contain proteins.
  6. Consider taking a photo of the gel rather than scanning. If you must scan the gel, sandwich the gel between two pieces of acetate (that have been washed in dH2O), and put the sandwich on a scanner.
  7. Cut out 2 control pieces: one blank area of the gel and one intensely stained area. The former will give you information on the level of keratin contamination and the latter on the efficiency of the digestion procedure. These are mandatory for each experiment.


Reagents and solutions

25 mM NH4HCO3 (100 mg/50 ml)
25 mM NH4HCO3 in 50% ACN
50% ACN/5% formic acid (may substitute TFA or acetic acid)

10 mM DTT in 25 mM NH4HCO3 (1.5 mg /mL). Make fresh.
55 mM iodoacetamide in 25 mM NH4HCO3 (10 mg /mL). Make fresh and protect from light.
12.5 ng/μL trypsin in 25mM NH4HCO3 (freshly diluted)

Gel fragment preparation

Excise protein bands. Cut each into 1 mm pieces. Place into a low-binding tube (LoBind, Eppendorf). Also cut out a gel piece from a protein-free region and an intensely stained region as controls.


Coomassie Stain and SDS Removal

  • Add 200 μL of 25 mM NH4HCO3/50% ACN and vortex for 10 min.
  • Using gel loading pipet tip, extract the supernatant and discard.
  • Repeat steps 3 and 4 until the gel pieces are colorless.
  • NOTE: Gel pieces can be left in supernatant overnight to avoid long de-stain times. Change supernatant until it no longer turns blue and then leave gel pieces to vortex overnight.
  • Remove supernatant (discard). Add 100% acetonitrile to cover the gel pieces. Let stand for a few minutes until the gel pieces shrink and turn white.
  • Remove acetonitrile (discard). Speed Vac the gel pieces to complete dryness (~10 min). 

Reduction and Alkylation

  • Rehydrate gel pieces in ~40 μL of 10 mM DTT (ensure full gel coverage). Vortex and spin briefly. Allow reaction to proceed at 56 °C for 45 min.
  • Remove supernatant and add 40 μl of 55 mM iodoacetamide to the gel pieces. Vortex and spin briefly. Allow reaction to proceed in the dark at room temperature for 30 min.
  • Remove supernatant (discard). Wash gel pieces with 100 μl of 25 mM NH4HCO3, vortex 10 min, & spin.
  • Remove supernatant (discard). Add 200 μL of 100% acetonitrile, vortex briefly, and let it stand for few minutes until the gel pieces shrink and turn white. If gel pieces do not look opaque, remove acetonitrile and repeat the step.
  • Remove the acetonitrile (discard). Speed Vac the gel pieces to complete dryness (~10 min).

Digestion

  • Add an excess of trypsin solution to completely cover gel pieces. This volume will vary from sample to sample, but on average 25 μL is sufficient.
  • Spin briefly and incubate at 37 °C, 4-16 hrs.


Extraction of Peptides

Spin down and aspirate supernatant (contains tryptic peptides) into Eppendorf tube.
Add 30 μL of 50% ACN/ 0.1% FA in water to the gel pieces, vortex 15 min., & spin. Aspirate supernatant and combine with the water extract taken in the previous step.
Speed Vac combined peptide extracts to dryness and resuspend in 20 uL LC buffer (0.1% formic acid, 2% acetonitrile in water).

References
Shevchenko et al. Anal. Chem.(1996) 68:850-858
Havlis et al. Anal. Chem. (2003) 75:1300-1306.