Guidelines for Sample Preparation and submission for LC-MS/MS analysis

The following are general guidelines for sample preparation and submission prior to LC-MS/MS analysis. Customers are strongly encouraged to follow these directions in order to ensure successful analysis and detection of their proteins of interest.

For further information, please contact the CAPA directly.

• Minimal amount of starting material:

  The following amounts of starting material are a general guide based on the sample media:

  Sample Type	Amount required
  2D-gel	400 µg.
  SDS-PAGE	40 µg.
  IP	50 fmol.
  In-solution	50 fmol.

• Detergents and surfactants:

  Although these reagents are good for solubilizing your proteins, their presence in the sample has adverse effects on the HPLC column and electrospray signal. Reagents such as SDS, Tween, NP-40, and Triton-X are not compatible with LC-MS/MS analysis, even in very low amounts (0.01% (v/v) of the total sample volume). Their removal prior to analysis is necessary; this can be accomplished by SDS-PAGE or another chromatographic separation (e.g. SPE, HPLC).

• 1D-gels:

  Please excise your gels bands and provide them to the laboratory in an Eppendorf tube without any liquid or buffer added. It is recommended to place one excised band per tube; multiples bands per tube could reduce the efficiency of protein digestion and peptide extraction.

• Immuno-precipitation (IP):

  If protein elution from your antibody is not possible, we recommend doing an SDS-PAGE separation to remove antibody fragments from your sample. Excess digested antibody will interfere in the detection of your protein of interest during LC-MS/MS analysis.

• Large-scale phosphopeptide enrichment:

  The minimum amount of protein starting material is 250 µg. Please perform cell lysis and determine the protein content of your sample prior to sample submission

• Mutations and variants:

  If your protein is not wild type, please provide us its complete amino acid sequence.

• Protein digestion:

  The enzyme we typically use for digestion is trypsin (modified, sequencing-grade); this will generate peptides cleaved at the C-terminal end of lysine and arginine. Please inquire first if a different enzyme is needed to digest your protein sample. An added cost may be included in the price if another enzyme is required for digestion.