Sample Analysis

Sample Submission

Below are several steps to follow if wish to have your samples analyzed by the facility personnel:

1. Identify the most suitable approach which will provide information you are looking for
2. Become familiar with sample preparation guidelines and make sure that the sample(s) meet the guidelines
3. Fill out a sample submission form
4. Deliver samples accompanied by the sample submission form(s).

It is suggested that we receive samples as solid material (just enough to see it) or neat liquid (several ul) for FAB, ESI, APCI. For MALDI-TOF analysis submit ~10 pmol of peptide, protein or oligonucleotide (>100 pmol) dry or in minimal (several ul) volume of solvent.

Sample Preparation

Whereas it is impossible to provide detailed instructions applicable to every aspect of using the facility, the following sample preparation information may be useful.

ESI sample requirements

Be sure to use only bottled HPLC grade water (no DI water of any kind!) and HPLC grade organic solvent such as methanol, acetonitrile, isopropanol. The water content may be anywhere in 10-90% range and usually governed by HPLC separation requirements. Organic acid such as acetic, formic and triflouroacetic (0.1-1%) is frequently used to promote positive ion formation. The later can form strong ion pairs with positively charged analyte(s), decreasing sensitivity. For this reason, TFA should be avoided in samples with a very low concentration of analyte(s).

Mass spectrometry is a very sensitive technique. For example, one can easily detect ppt levels of perch lor ate ions in negative ion mode and low ppb levels of triethylamine looking at positive ions. Most real world samples contain salts, buffers and other compound of concern. Compounds of special concern can be divided as follows:

Salts

The sample must not contain any salts such as NaCl, K₂HPO₄, etc. The positive ions (Na⁺, K⁺ , Ca²⁺) can substitute for H⁺ in the analyte giving rise to adduct ions which greatly complicate interpretation of the spectra and degrade sensitivity. Negative ions (Cl^-, SO₄^2-) can form ion pairs with basic sites and neutralize positive charge in the analyte(s). Furthermore, positive salt ions can be reduced in the mass spectrometer and plate out on the ion optics, damaging the instrument. If your separation protocol uses a buffer, replace it with a volatile ammonium salt such as acetate and bicarbonate. In LC-MS experiments salt contamination is rarely a problem. For direct infusion use an appropriate desalting technique.

Buffers

Be sure the sample does not contain any organic buffer salts such as TRIS, MES, MOPS, etc. for the same reasons listed above. This also includes reducing agents such as BME and DTT

Stabilizers and detergents

Glycerol, PEG, ionic (e.g. SDS) and non-ionic (e.g. Triton X-100) detergents are compounds of a special concern in ESI MS. These components ionize particularly well, especially PEG, and if they are present, that is all you will see!

Sample preparation for MALDI experiments

The key to success for a MALDI analysis is to produce a thin layer of analyte ultimately mixed with UV absorbing compound (matrix). A short description of basic steps involved in MALDI sample preparation can be downloaded here.

In general, MALDI as a technique is more tolerant to sample impurities. However levels of buffers and detergents which exceed the following limits will probably cause noticeable degradation of spectra: