Proteomics Core Facility

Recommended basic experiment

• Check the quantity and complexity of your protein sample by running an SDS-PAGE gel and examining the banding pattern after silver stain. The appearance of visible silver stain bands generally indicates there is enough protein for identification. Gel pieces can be cut from silver stained gels, stored in 1% acetic acid, and submitted to the Core Facility for analysis. It is recommended to send the Core Facility a picture of your gel, with the planned cutting areas marked, for review before cutting out your bands.

Quantity

• Gels: visible bands on a silver stained gel generally indicate there is enough protein for identification.

• Affinity Purification/MS: 1-5 ug of protein per lane is a suggested starting point.

• Whole Proteomes: 25 ug total protein per lane is a suggested starting point.

• Solutions: 50 - 500 femtomoles of the most abundant protein in the mix.

• The above are suggestions for typical samples. Significantly lower amounts are detectable under ideal conditions.

• The nanoLC-MS/MS system is a highly sensitive precision instrument; sample purity is more important than amount.

Contamination

• Contamination is a major issue that can negatively affect the performance of the LC-MS system!

• All proteomics samples must be free of PEG (polyethylene glycol) or PEG-like chemicals.

• Potential sources of these chemicals are soaps, detergents, lotions, low-quality plastic labware, or improperly cleaned glassware. All proteomics-related sample prep should be done with acid-washed glassware or alcohol-washed plastic that is of high quality and suitable for proteomics work. Soap/detergent washing should be avoided as much as possible and anything that might have come into contact with soap should be rinsed extra-thoroughly. Any sort of hand creams, greases, lubricants must be avoided as they will contaminate not only your sample but also other samples that are processed afterwards.

• Avoid contaminating your samples with irrelevant proteins such as keratin (skin, hair, wool clothing), albumin (cell culture serum, blocking solutions), milk proteins (blocking solutions), immunoglobulin (IP reagents), protein A/G (IP reagents), or synthetic peptides (elution or blocking peptides).

Sample submission

• Please label all tubes clearly with a distinctive identifier such as your name and the date.

• Please fill out the Service Request Form completely and correctly.

• For gels, it is strongly recommended to provide a picture of your gel with the submitted bands indicated.

Additional information

Stains

• Mass spec compatible silver (suggested protocol listed below or Shevchenko 1996), coomassie blue, or sypro stain are acceptable.

Gel bands

• Use only the cleanest high-quality materials when preparing your gel.

• Do not allow gels to dry out or crumble; particles from damaged gel can block the nanoLC system.

• When cutting out the gel region(s) to be analyzed, submit only the main part of the band or lane. Do not include lane margins, dye fronts or stacking gels.

• Submitted gel pieces must be no larger than 10 mm x 10 mm, per sample. Darkly stained gel pieces should be no larger than 10 mm x 2 mm.

• Store excised gel bands in 1% acetic acid.

Solutions

• All in-solution samples must be of the highest purity. Samples must be free of both physical and chemical contaminants. In-solution samples cannot contain detergents.

Mixtures

• Hundreds of proteins may be identifiable, depending on the sample. Highly-abundant proteins can block the detection of lower-abundance ones.

Urea

• Heating samples in urea is not recommended.

Non-identifiable proteins

• The standard protein identification workflow uses trypsin to digest proteins into peptides, and uses a reference database for protein identification. Therefore the following will not be identified:

  • Proteins not listed in the reference proteome database.

  • Tagged or mutated peptides (only the peptide bearing the tag or mutation is affected).

  • Proteins with no (or too few or too many) trypsin cleavage sites (other proteases can be substituted).

  • Peptides (post-digest) smaller than 6 amino acids or longer than about 30 amino acids.

  • Peptides with unexpected post-translational modifications.

Post-translational modifications (PTMs)

• Biological PTMs such as phosphorylation are not checked for during data analysis unless specifically requested.

• Some PTMs require special sample preparation or detection methods.

• Please note on your service request form any modifications introduced experimentally (labels, cross-linkers, iodoacetamide or iodoacetic acid treatment, MMTS treatment, tags or mutations, etc.).

Hazards

• Hazardous samples (radiation, biohazards, toxins, bio-active peptides, live viruses or organisms) cannot be accepted by the Core Facility.

Consultations

• Consulting with the Core Facility before beginning your experiment is highly recommended.

1. Purpose 

   1. This protocol describes the excision of stained protein spots/bands from a polyacrylamide 
      gel. The excised gel can be submitted for protein identification by mass spectrometry.

2. Reagents and Materials

   1. 2.1. 1.5 ml (or 0.5 ml) microcentrifuge tubes, siliconized (or low protein-binding)

   2. 2.2. Acetic acid

   3. 2.3. Scalpel with fine-sized blades (e.g. #11 surgical blade)

   4. 2.4. Clean work area (e.g. positive pressure clean hood or enclosed laminar flow hood)

   5. 2.5. 90% ethanol

   6. 2.6. 70% ethanol

   7. 2.7. Water (Milli-Q or Optima grade, or similar)

   8. 2.8. Suitable cutting surface (e.g. light box, glass plate), very clean

   9. 2.9. Clean, non-latex gloves

3. Reagent Preparation

   1. 3.1. 1% Acetic Acid

   2. 3.1.1. Add 10 μl of glacial acetic acid to 990 μl water (assume 1 ml per 10 gel bands).

   3. 3.1.2. Vortex to mix.

   4. 3.2. Clean tubes

   5. 3.2.1. Clean the microcentrifuge tubes with 90% ethanol (or methanol) and dry 
      completely.

4. Procedure

   1. 4.1. Thoroughly clean the cutting surface with 70% ethanol (or methanol) and place it into 
      the clean work area.

   2. 4.2. With a clean blade, excise the protein band or region of interest.

   3. 4.2.1. If cutting a single band (rather than a multi-band region), do not include any nonstained polyacrylamide. If the band is large, excise only the darkest central portion.

   4. 4.2.2. Push the blade straight down to avoid creating rips or tears.

   5. 4.3. Place the cut gel into a pre-cleaned microcentrifuge tube and add sufficient 1% acetic 
      acid to cover the gel (if commencing an in-gel digest immediately, water can be used 
      instead).

   6. 4.4. If cutting a large number of spots, add water to the gel as needed to prevent drying. A 
      squirt bottle filled with milli-Q water is handy for this purpose. Dry gel will curl and 
      may rip. 

   7. 4.5. Store at 4C for short-term or freeze for long-term.

5. Notes

   1. 5.1. Extreme care must be taken to avoid contaminating the gel with keratin (the major protein of skin and hair) or other environmental proteins. A lab coat with elastic or fitted cuffs should be worn. Wool clothing should not be worn. Clean non-latex gloves must be worn at all times. Prior to cutting, the gel should be kept covered at all times. 

   2. 5.2. Use only the highest-purity reagents for proteomics experiments. Commercially precast gels are often cleaner than lab-made gels.

   3. 5.3. If the equipment or glassware used to run the gel is also used for Western blots, take great care to avoid cross-contamination from Western blotting reagents, especially “blocking” proteins such as skim milk powder or serum albumin.
       

Note:
The following research protocol is intended for use only by qualified personnel in a 
properly equipped laboratory and who have been trained in the safe handling of 
chemicals. 
Please dispose of all chemicals safely and in accordance with applicable regulations.

Silver Stain for Polyacrylamide Gels (Based upon Shevchenko et al. Analytical Chemistry (1996) 68:850-858)

Reagents:

• Fixer: 50% ethanol, 5% acetic acid, in water 

• Wash: 50% ethanol in water 

• Sensitizer: 0.02% sodium thiosulfate 

• Stain: 0.1% silver nitrate, in water 

• Developer: 0.04% formalin in 2% sodium carbonate 

• Stop: 5% acetic acid in water 

• Storage: 1% acetic acid in water 

Procedure: 
a) 30 minutes in Fixer (can be left longer) 
b) 10 minutes in Wash 
c) 2 × 10 minutes in water 
d) 2 minutes in Sensitizer 
e) 2 × 3 minutes in water 
f) 30 minutes in Stain 
g) 1 minute in water 
h) add a small amount of Developer, swirl briefly and discard 
i) add more Developer and shake slowly until spots/bands appear 
j) 5 minutes in Stop solution 
k) store in Storage solution at 4° 

Destain Procedure (Based upon Gharahdaghi et al. Electrophoresis (1999) 20:601-605)
(Optional: if staining gave poor results, you can destain and try again) 

Reagents: 

• Solution A: 30 mM potassium ferricyanide 

• Solution B: 100 mM sodium thiosulfate 

• Destain: mix solution A and solution B in a 1:1 ratio just before use 
   

Method: 
a) Immerse the gel in Destain 
b) Brownish colour should be removed within 3-5 minutes 
c) Wash 3 × 10 minutes with deionized water or until yellow colour is removed

Tips For Affinity-Purification (‘Pull-Down’) Proteomics 

The most common application of proteomics to cell biology and related research involves 
a ‘pull-down’ assay to determine the interaction partners for a protein of interest. Pulldown may be achieved by various methods (immunoprecipitation, TAP tags, in vivo
biotinylation, etc.). The following tips may help optimize your experiment.

• Choose an appropriate negative (background) control. Remember that some proteins will stick to beads, antibodies and/or plastic surfaces in a non-specific way. If available, a cell line with knock-down or deletion of the protein of interest makes an excellent negative control. 

• Optimize your stringency. Experiment with different salt concentrations to find an optimum balance between reducing background and maintaining bona fide low-affinity interactions. 

• Use a thin polyacrylamide gel. Unless you need to accommodate large amounts of protein, extra gel only contributes extra background. 

• Keep your sample free of irrelevant environmental proteins. Keratin (the major protein of skin and hair) can enter your reagents at any stage. Keep everything clean, from the stock reagents onwards. 

• If applicable, isolate only the subcellular region where your complex of interest resides (e.g. nucleus). 

• If using immunoprecipitation, cross-link your reagents to reduce the amount of antibody in the final sample. If possible, affinity-purify your antibody before use. Selective elution (e.g. competitor peptide) can potentially increase the specificity of your pull-down. 

• Try pre-clearing your sample (e.g. for an immunoprecipitation, pre-incubate with beads but without antibody). If using a centrifugation-based pull-down, pre-clear your sample of non-specific precipitators with a centrifugation step prior to performing the pull-down. 

• Try different bead types (e.g. magnetic, sepharose, agarose) to see what gives the lowest background for your application. 

• Do not over-centrifuge beads or subject them to physical damage. 

• Pre-wash sample tubes with ethanol, methanol or other organic solvent to ensure they are protein-free.