for sample preparation are provided below
contact us with
specific questions regarding your particular sample(s).
& SMALL MOLECULES
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
- Please specify
if particular solvents should not be used.
- 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.
- 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.
of non-volatile components should be kept below 1 mM, if not eliminated
- Volatile buffers
such as ammonium acetate and formate are tolerable below 20 mM.
- Glycerol should
be limited to no greater than 1%.
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
- LC-MS analysis may be utilized for on-line sample cleanup and concentration.
Proteins in polyacrylamide
- 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
- 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
- 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.