Sample Guidelines

Sample submission guidelines

APM reserves the right to refuse samples that do not meet facility conditions. Poorly prepared samples can damage equipment and create significant costs in time and resources.

Pre-submission consultation

If you are unsure how to prepare mass spectrometry-ready samples, contact Jack Moore at jmoore@ualberta.ca or 780-492-2522. He can assess your needs, answer questions, or arrange a consultation with a faculty member.

Submitting samples

First-time users should arrange a meeting with Jack at APM to review the submission process. If you drop off samples without a meeting, email a brief overview of your preparation and analytical goals. Please submit sample information prior to mailing using the form below.

Naming samples and sample tubes

• Keep names as simple as possible, 3 digits or characters maximum.
• If you supply us with a naming key we will label your results with a longer name(s) once our analysis is complete.
• Label both the top and side of the tubes.
• If you are submitting a number of sample groups with replicates, name the samples so that the groups, and replicates within the groups, can be easily deciphered.
• Microcentrifuge tubes are the only acceptable option.
• Use snap cap tubes, no screw tops.

Shipping samples

• Ship samples frozen on dry ice.
• Email the tracking number to Jack so delays can be monitored.
• Include a sample submission form or email it to Jack.
• Use overnight shipping. FedEx is preferred.

Sample preparation guidelines

Avoiding contamination

Keratin from dust, skin, or hair, along with PEG, detergents, high salt concentrations, and many polymers, can reduce or prevent usable data. Review our contamination-minimization guide.

On-bead sample preparation

• Samples must be free of detergents (SDS, Triton, Tween, etc.), and higher concentrations of glycerol and polymers such as PEG.
• It is important that the antibody, or other compounds, used as bait are cross-linked to the beads.
• The bead/protein complex should be washed a minimum of 4 times using dilute ammonium bicarbonate, 25 – 50mM, or 1x PBS.
• Remove liquid after washes and freeze magnetic beads before submission.
• Do not freeze agarose beads. Store at 4C and drop off as soon as possible (contact us to arrange)
• Estimate the amount of protein being submitted using your input and antibody capacity.
• An immunoblot signal is not an indication that the MS procedure will be successful.

In-solution or dried pellet sample preparation

• Samples must be free of detergents (SDS, Triton, Tween, etc.), and higher concentrations of glycerol and polymers such as PEG.
• Supply an exact account of everything that is in the sample buffer, or was in the buffer before drying.
• Perform a quantification assay before submission. Make sure that the chosen assay is suitable for your sample type.
• Freeze samples at -80C before submission.

Lysate submissions for DIA analysis sample preparation

• We recommend using a RIPA buffer (Thermo 89900, and optional + 2% SDS). Urea (6–8 M) in 50–100 mM Tris-HCl or TEAB (pH 8.0–8.5) also works.
• On-campus clients can purchase the buffer from the BioChem stores at 354 Medical Sciences Bldg.
• Submit 30-50ug or total protein per sample (most DIA injections use 200 ng to 1 µg of digested peptides, but extra input ensures accurate quantification and backup capacity).
• Biological replicates are highly recommended and we recommend 4 replicates as the minimum. DIA relies heavily on statistical consistency across runs to quantify thousands of proteins.
• Wash cell pellets 2–3 times with ice-cold 1X PBS before lysis to clear culture media components and fetal bovine serum (FBS).
• Include a fresh Protease Inhibitor Cocktail (EDTA-free) during the initial lysis step.
• Perform sonication or high-speed shearing to clarify genomic DNA, then centrifuge at high speed (14,000 g) to pellet insoluble debris.
• Quantify the protein concentration using a BCA or Bradford assay before submitting or performing digestion.
• Snap-freeze clear lysates in liquid nitrogen or on dry ice, and store at -80°C in clear Eppendorf Protein tubes (LoBind if possible). Avoid freeze-thaw cycles.

In-gel sample preparation

• Supply as much protein as practical to maximize the likelihood of ideal data.
• Concentrate protein into the smallest practical gel volume. For non-complex samples, run the gel only about 1 cm.
• Use a 1 mm gel thickness when possible.
• Use Coomassie blue and stain only long enough to visualize the bands.
• Destain thoroughly until the background is clear.
• Cut closely around stained areas and exclude unstained gel.
• Pool equivalent bands from multiple lanes into one tube.
• Wash gel slices twice with 50% acetonitrile in water, then add only enough water to keep them moist.
• Store frozen at -20°C until submission.

MALDI-TOF sample preparation

• Use volatile buffers and solvents such as water, methanol, or acetonitrile.
• Avoid non-volatile salts, detergents, urea, guanidine, glycerol, DMSO, and DMF. ZipTips or reversed-phase LC may help remove them.
• Ideal starting samples are 5 µL at approximately 15 pmol/µL for MW 1,000 or less, 50 pmol/µL for MW 20,000, and 100 pmol/µL for MW 75,000.