We highly recommend discussing the project(s) with PRF personnel before preparing the samples.
Contact the Laboratory Director and/or Manager to discuss the project before preparing the samples.
The turn-around time is generally 2 weeks but can vary depending on the complexity and nature of the analyses proposed.
Attn: Venkatesha Basrur, Ph.D.
MSRB II, Room 3556
1150 W. Medical Center Dr.
Ann Arbor, MI 48109
General guidelines for a successful proteomic analysis of your samples
Minimize the contaminants: The PRF uses state-of-the-art, highly sensitive mass spectrometers to fulfill its mission. It is worthwhile to note that while the PRF strives hard to obtain the best results possible, the quality of the data generated by the mass spectrometer is only as good as the quality of the samples introduced into them. Hence it is imperative that the samples intended for MS analysis be prepared by taking extreme care to avoid some of the common contaminants which can impair an otherwise well planned and executed experiment. These contaminants are either “chemical contaminants” or “exogenously introduced protein” contaminants.
Chemical Contaminants: Polymers (from plasticware), buffer components (i.e. inorganic salts, detergents), protein stabilizing agents (i.e. glycerol, PEG). While inorganic salts are easily removed by reverse phase-based sample clean-up steps, other contaminants persist even after.
- To minimize polymer contamination, please rinse plastic ware with HPLC grade organic solvent (Methanol or Acetonitrile), if available.
- For biochemical techniques (cell/tissue lysis, IP, affinity purification, etc.) use the least complex buffer required. Some of the commonly used buffers such as RIPA containing small amounts of detergents (ex. 0.1% triton, 0.1% SDS etc) are OK.
- Use the highest quality chemicals/reagents available.
- Please consult with the Laboratory Manager before you begin the experiment to verify the MS compatibility of your buffers components.
Exogenously Introduced Protein Contaminants: Mass spectrometers, under normal operational conditions, are not quantitative (Signal intensity depends on various properties of the peptide/protein including the ionization efficiency). Hence contaminating proteins which may ionize more efficiently and/or co-elute with the peptide of interest can drastically affect the out come of an experiment. Most common contaminants observed are cytokeratin (coming from skin and hair follicles) and serum proteins (i.e. albumin).
- Wear gloves all the time and change them often.
- If possible, keep a small area just for MS sample processing. Wipe the area clean with methanol or ethanol.
- Keep apparatus (staining trays, electrophoresis unit, pipettes, etc.) exclusively for MS sample preparation whenever possible.
- Wash the cells/tissues extensively with isotonic buffer BEFORE lysis (to remove any serum proteins coming from tissue culture medium or blood)
- Use pre-cast gels.
- Handle the gel as little as possible and keep it covered at all times. Consider using StainEase staining trays or equivalent.
- Run a parallel sample for documentation purposes, if possible.
MS compatible protein staining: The following protein stains have provided satisfactory results in our hands.
- Coomassie stain: Any single-step Coomassie stain that doesn’t require extensive and multiple changes of destaining solution works well. We recommend Colloidal Blue or SimplyBlue stains.
- Silver stain: Classical silver stain techniques that utilize glutaraldehyde and/or formaldehyde are not suitable as they modify amino acid side chains and interfere with digestion with protease. Several MS compatible silver stains are commercially available. We recommend ProteoSilver or SilverQuest kits. Please remember to bring the appropriate destaining solutions when submitting samples.
Guidelines for excising Coomassie or MS-compatible silver stained gel slices: (If you are located on or near the Ann Arbor campus, you can bring the whole gel to our laboratory and we will cut the gel slices for you.)
- Please use 1 mm thick gels whenever possible.
- Follow either Coomassie or MS-compatible silver staining protocols.
- Once the band/spot of interest has been located, using a clean scalpel blade, cut the gel as close to the stain as possible.
- If you are planning to process the whole lane, transfer the gel onto a clean glass plate and cut the lane into 12-16 equal sized slices (~0.5 cm each)
- Using a clean pair of forceps, transfer the gel slice(s) into a 1.5 ml eppendorf tube. There is no need to include any buffer/water in the tube.
- This can be kept at -20°C until submission for analysis. If shipping from outside, ship it overnight on dry ice.
- Please ensure that properly filled sample submission forms accompany every sample.
Guidelines for submitting samples for In-solution digestion: As a general rule, it is safe to assume that many of the commonly used biochemical reagents, especially detergents, may interfere with MS analysis. Please discuss with the Laboratory Director and Manager before preparing the samples.
Guidelines for submitting samples for On-bead digestion (ex. For interactome analysis): Please discuss with the Laboratory Director and Manager before preparing the samples.
In most cases, Investigator can follow their optimized protocol for sample preparation. However, after the final wash, we request you to perform 1-2 brief rinses with a simple buffer (ex. PBS or TBS) to remove any residual detergents or protease inhibitors etc. After rinsing, remove all buffer solution and submit just the beads for MS analysis. You may store the beads, after removing buffer solution, at -80 C till you submit.
Other points to consider:
- Use magnetic beads. This format is very useful to remove the final wash buffer almost completely before freezing the beads.
- Use antibody coupled beads, if available.
- If you believe that the final wash with a simple buffer might disrupt your interactome, please discuss with the Lab Manager about the buffer compatibility.
Guidelines for submitting samples for PTM analysis: Please discuss with the Laboratory Director and Manager before preparing the samples.
- Use appropriate inhibitors, when available, for sample preparation in order to make sure that the sample is enriched for PTM of interest (ex. phosphtase or proteosome inhbitors).
- As a general rule, PRF will require higher quantities of protein in appropriate buffers for PTM analysis.