Sample Preparation Guidelines

  • 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
  • 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