Shipping Address
Attn: Venkatesha Basrur, Ph.D.
MSRB II, Room 3556
1150 W. Medical Center Dr.
Ann Arbor, MI 48109
Phone: 734-615-5722
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.
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).
MS compatible protein staining: The following protein stains have provided satisfactory results in our hands.
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)
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:
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.