FAQs & SOPs
Frequently Asked Questions (FAQs)
What precautions be taken during sample preparation?
- Wear gloves at all times, use clean reagents and glassware to avoid keratin contamination!
- Sample prep for SDS-PAGE: Avoid boiling samples, if possible - heating at 37˚C for 15 min prior to electrophoresis is normally sufficient. If urea must be used (eg. preparation of IEF samples for 2D-PAGE analysis), never heat sample above 37˚C. Let us know if any chemical modification (eg. iodoacetamide to block sulfhydryl groups) has been used in sample preparation.
- Protein stains: Most gel stains for proteins (eg. Coomassie Blue, GelCode, sivler staining) are compatible with MS analysis, but do not use silver stains that include covalent fixatives (ie. glutaraldehyde). If feasible, provide gel slices containing at least 1 pmol of sample (eg. 0.1 µg of 100 kDa protein), which is normally near the limit of Coomassie Blue detection. While identification is possible with lower amounts (~100 fmol), the success rate drops considerably. Do not store gels in glycerol.
- Gel slices. This is the most critical stage to avoid contamination - use clean materials and work in a laminar flow hood wherever possible!! Using a clean scalpel, cut out each protein band. Gel slices should not be wider than a 2 or 3 millimeters. Please cut exactly around or within the stained protein to avoid excess gel. The protein should be focused on as small a piece of gel as possible. Transfer to a microfuge tube (do not mash or cut into finer pieces). Sterile 1 ml disposable pipette tips can also be used to excise 2D spots. Gel slices should be cut as soon as possible and stored in a -80˚C freezer prior to analysis.
- Detergents: Certain detergents can give rise to a polymer-like background and cause severe signal suppression during LC-MS acquisition. Avoid detergents such as Tween-20 and Triton X-100 if possible. Due to low protein solubility the use of detergents may be unavoidable. Look for MS friendly detergents and inform us whenever nonionic detergents are used.
In what conditions should samples be shipped?
In most cases, freeze dried/lyophilized samples (eg. protein extracts) may be shipped in sterile plastic tubes (preferably 1.5 or 2 mL tubes) at room temperature. Gel bands may be dried after excision in a vacuum centrifuge.
Fresh samples can be refrigirated or frozen and shipped with ice packs or dry ice, accordingly.
How and when do I submit my samples?
Please contact email@example.com to arrange a sample delivery date.
All samples sent within Dalhousie University should be properly labeled and dropped off in the designated and labeled fridge located in the CORES hallway (LSRI, outside Room N109).
Samples may also be sent by courier* to the following address:
Att: Dr. Alejandro Cohen
Biological Mass Spectrometry Core Facility. Room N105
Life Sciences Research Institute (LSRI)
1348 Summer St. (Receiving)
Halifax, NS B3H 0A8
*We recommend sending frozen/refrigerated samples early in the week (Monday-Tuesday) by express (24-48hs) delivery to avoid weekend delays.
How much will my analysis cost?
The cost of analysis will depend on the type and number of analyses performed. View a detailed description of pricing or contact firstname.lastname@example.org for specialized and custom-made analyses. Samples submitted by external Academic institutions and Industry receive an additional 25% and 50% surcharge, respectively, on their final invoice.
Are services only available to external (Non-Dalhousie) clients?
Priority is given to Dal researchers, however samples from external clients (Industry, Academia, Government Departments, NGOs etc) are also accepted depending on the instrument availability. Samples submitted by Academic institutions and Industry receive an additional 25% and 50% surcharge, respectively, on their final invoice.
How long do the analyses take?
SDS-PAGE Gel bands or spots for protein ID should be submitted in the dates specified in Services. Reports are usually ready two or three weeks later. Gel bands received after the submission dates are stored at -20C until the next batch, the following month.
For other analyses, the duration of analysis depends on the amount of sample prepping required, the complexity of each analysis and the current workload which varies from month to month. Typically, results are reported anywhere between 1 week to 3 weeks after sample submission.
How are results reported?
Protein identification from Proteome Discoverer Software is typically reported in user friendly Excel files. The filenames are structured as:
YYMMDD corresponds to the date of MS aquisition
AAAA: Request/Invoice number
BBB: Researcher's ID number
S##: Sample ID number as entered in the Request Form.
The Excel files (.xlsx) contain all the information generated by ProteomeDiscoverer software. The information is nested into Protein Groups, Proteins and Peptides tables. Each one of these tables can be exposed by clicking on the '+' signs found on the left hand side of the spreadsheets (Columns 1, 2 and 3). The identifications scores, peptide sequences, PTM modifications and complementary data are found in columns within each nested table.
Important reminder: Bottom-up proteomics relies on the digestion of proteins into peptides, usually done with trypsin. When the digests are analyzed by LC-MS/MS, the data collected is used to identify the peptides using data base search algorithms. Based on these peptide hits, Protein identifications are inferred. Proteins are grouped together (Protein Groups) when there is insufficient evidence to claim the presence of any single proteoform, and is quite common with proteins that share many digested (tryptic or other enzyme) peptide sequences.
The proteins within a group are ranked according to the number of peptide sequences, the number of PSMs, their protein scores, and the sequence coverage. The top-ranking protein of a group is labeled as the 'master protein', and appears as the Protein Group 'representative'.
The reports can also be explored using Proteome Discoverer software in the PCF lab with support from our staff. Contact us at email@example.com to arrange an appointment.
Alternatively, .raw, .msf (Thermo’s Mass Spec Format files), .wiff (ABSciex files), .mgf, .mzML and .mzXML can be provided upon request via Dalhousie's File Exchange server.
Standard Operating Procedures (SOPs)
1. Fix the gel slab in 50% methanol, 5% acetic acid in water for 20 min.
2. Wash for 10 min with 50% methanol in water and additionally for 10 min with water to remove the remaining acid.
3. Sensitize gel by a 1 min incubation in 0.02% sodium thiosulfate, and then rinse with two changes of distilled water for 1 min each.
4. Stain: submerge gel in chilled 0.1% silver nitrate solution and incubated for 20 min at 4 °C. After incubation, discard the silver nitrate, and rinse twice the gel slab with water for 1 min
5. Develop the gel in 0.04% formalin [35% formaldehyde in water] in 2% sodium carbonate with intensive shaking. After the developer turned yellow, discard and replace with a fresh solution. It is essential that the developing is carried out in an absolutely transparent solution. After the desired intensity of staining is achieved, the development is terminated by discarding the reagent, followed by washing of the gel slab with 5% acetic acid.
Silver-stained gels were stored in a solution of 1% acetic acid at 4 °C until analyzed.
* Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels
Andrej Shevchenko, Matthias Wilm, Ole Vorm, and Matthias Mann
Anal. Chem. 1996, 68, 850-858
Protein Precipitation Techniques
Note: All steps should be carried out in glassware or solvent-compatible plasticware.
Acetone Precipitation Protocol
1. Cool the required volume of acetone to -20°C.
2. Add four times the sample volume of cold (-20°C) acetone to the tube.
3. Vortex tube and incubate for 60 minutes at -20°C.
4. Centrifuge 10 minutes at 13,000-15,000 x g.
5. Decant and properly dispose of the supernatant, being careful to not dislodge the protein pellet.
Optional: If additional cycles of precipitation are necessary to completely remove the interfering
substance, then repeat steps 2-5 before proceeding to step 6.
6. Allow the acetone to evaporate from the uncapped tube at room temperature for 30 minutes. Do not over-dry pellet, or it may not dissolve properly.
7. Resuspend in appropriate buffer.
TCA Precipitation Protocol
1. Add an equal volume of 20% TCA (trichloroacetic acid) to protein sample.
2. Incubate 30 min on ice.
3. Spin in microfuge at 4 deg. For 15 min.
4. Carefully remove all supernatant.
5. Add ~300 ul cold acetone and spin 5 min at 4 degrees.
6. Remove supernatant and dry pellet.
7. Resuspend samples in desired buffer.
Warning: TCA is a strong acid and should be handled with care
1. To sample of starting volume 100 ul
2. Add 400 ul methanol
3. Vortex well
4. Add 100 ul chloroform
6. Add 300 ul H2O
8. Spin 1 minute @ 14,0000 g
9. Remove top aqueous layer (protein is between layers)
10. Add 400 ul methanol
12. Spin 2 minutes @ 14,000 g
13. Remove as much MeOH as possible without disturbing pellet
14. Speed-Vac to dryness
Reference: Wessel, D. and Flugge, U. I. Anal. Biochem. (1984) 138, 141-143