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Guidelines for sample preparation are provided below

Please contact us with specific questions regarding your particular sample(s).


Whenever possible, sample should be submitted as solids, along with the appropriate solubility information. This method is especially recommended for samples which are sent by mail. For most compounds, microgram amounts are sufficient; milligram amounts are preferred. Excess sample which is not consumed in the analysis can be returned to the investigator. On-campus researchers are requested to pick up their samples after analysis.


  • In general, volatile, low MW protic solvents are preferred.
  • Methanol is the default solvent; acetonitrile is a good alternative.
  • If water is required for solubility, up to 50% water may be added.
  • High MW or viscous solvents should be avoided, including DMSO, DMF, and THF.
  • Hydrocarbon solvents, such as hexane and benzene, are not amenable to ESI.
  • Acetone may be used but is not preferred, as even high-grade acetone typically contains contaminants which show up as strong peaks in the MS, and may overwhelm the analyte signal.
  • If the sample contains TFA (e.g. from a reverse-phase HPLC run), remove the TFA by lyophilization or by drying down the sample overnight under high vacuum.
  • Please specify if particular solvents should not be used.

Concentration & Volume

  • High quality spectra can typically be obtained from 100 microliters of sample at 20-50 micromolar concentrations (20-50 micrograms/mL for a compound of MW 1000).
  • A reasonable estimate of sample concentration is important, as both extremely low and extremely high concentrations have detrimental effects.


  • Be sure to indicate if the compound is sensitive to acid or basic conditions, as small amounts of acid (formic, acetic) or base (ammonium hydroxide, triethylamine) are often added to samples in order to enhance ionization.
  • For example, a compound containing carboxylic acid groups may be dissolved in a slightly basic medium in order to deprotonate the carboxylic acid groups and maintain the compound in solution as its negative ion. This sample would be run in negative ionization mode.


Concentration & Volume

  • For routine intact mass analysis, the minimum amount of protein required depends on the MW of the peptide or protein.
  • Good results have been obtained with 25 pmol at 5 kD, 100 pmol at 20 kD, 200 pmol at 40 kD, and 500 pmol at 60 kD.
  • Sample concentration should be such that the appropriate amount of protein is contained in 20-25 uL.

Salts & Buffers

  • Salts and non-volatile buffers suppress ionization and can form adducts which affect the homogeneity of the analyte.
  • Concentrations of non-volatile components should be kept below 1 mM, if not eliminated completely.
  • Volatile buffers such as ammonium acetate and formate are tolerable below 20 mM.
  • Glycerol should be limited to no greater than 1%.
  • Trifluoroacetic acid (TFA) also causes signal supression, and should be kept below 0.1%. Commonly used alternatives to high concentrations of TFA are mixtures of either 1% acetic or 0.1% formic acid with 0.025% TFA.
  • The salt and buffer limits become increasingly stringent as the MW of the protein increases. To maximize the likelihood of a successful analysis, proteins greater than 40 kD ideally should be submitted in deionized water only.
  • LC-MS analysis may be utilized for on-line sample cleanup and concentration.


Proteins in polyacrylamide gel

  • Samples for protein identification by proteolytic digestion and LC-MS/MS are commonly submitted as Coomassie stained bands or spots in polyacrylamide gels.
  • The gels should be handled as little as possible, to minimize contamination by dust and keratin.
  • Destain Comassie stained gels by soaking for at least 2 hours in 10% acetic acid, 50% methanol, and 40% H2O with at least two changes of this solvent. If the gel still has a Coomassie Blue background then continue destaining until the background is nearly clear.
  • Sypro Ruby and other fluorescent stained gels should not require an additional destaining step.
  • After destaining, soak in pure water until pH is neutral.
  • The spot or band of interest should be excised cleanly, excluding all of the surrounding blank gel; the goal is to maximize the ratio of protein to gel.
  • Cut the excised gel into small pieces (1-2 mm square) and placed in a clean eppendorf vial.
  • Wet samples should be shipped on dry ice. Dry gel pieces may be shipped at room temperature.

Proteins in solution

  • Solution samples of proteins for identification will be treated with 8M urea to denature the protein before reduction and alkylation.
  • Samples should be shipped on dry ice.

Protein-related projects: MudPIT, de novo sequencing

  • Please contact SUMS to discuss sample prep and strategy for more complex projects such as MudPIT, peptide mapping, de novo peptide sequencing, and protein folding.


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Last Modified 6.27.06