Chemistry Reference
In-Depth Information
7.8.3
Sample Preparation and Limits of Detection
Sample 'contaminants' from an ESI-MS viewpoint include nonvolatile buffers, salts and
detergents, all of which may stabilize the target biomolecule sample but are incompatible
with ESI. These sample additives, through various mechanisms, can reduce or suppress
completely the ion abundance from the target of interest, leading to a poor or even null
result in terms of observation of the mass spectrum. Volatile solution components that
do not form gas-phase adducts with the target biomolecule are therefore essential and the
most investigated and proven reliable volatile components for this purpose include the salts
ammonium acetate and ammonium bicarbonate.
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These salts can be purchased as ESI-
MS grade, i.e. free of contaminants not compatible with ESI-MS. Proteins are generally
dissolved in pure water with ammonium acetate or ammonium bicarbonate present in
a concentration between 5 and 500 mM, depending on the protein. In-line techniques
such as desalting and size-exclusion chromatography have been routinely employed as a
sample cleanup directly prior to ESI-MS analysis. This permits target sample solutions to
be first exchanged into ESI-compatible buffers, replacing unwanted nonvolatile solution
components while preserving target-ligand noncovalent complexes. The importance of
these sample preparation considerations cannot be overstated and are probably the single
most common reason why, in the hands of the inexperienced MS operator, experiments
with native state proteins fail.
Modern ESI-MS instruments have sufficient sensitivity to detect proteins present at low-
and sub-micromolar concentrations. The most common types of ESI sources employed use
a pressurized pneumatic assist gas to assist in the formation of fine droplets by pneumatic
nebulization and operate with flow rates around 1 L min
−
1
. Thus, a screen can be carried
out with as little as 1 L of a protein at a concentration of about 1 M, meaning that as
little as a few picomoles of protein are sufficient for a screen. A number of different types
of ESI sources, known as nanospray sources, have been designed that can operate at even
lower sample flow rates (10-200 nLmin
−
1
). These generate smaller sample droplets and
improve the signal intensity of the protein-ligand noncovalent complexes further, with
the added benefit of reducing protein consumption up to 100-fold compared with standard
electrospray flow rates. Nanospray has also been reported to be more tolerant of nonvolatile
cations in solution.
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Nanospray has long been associated with difficult manual work
involving fine spray needles and is therefore avoided by many MS operators. Recently, an
automated fabricated chip nanospray source has been developed. This chip-based device
has improved the ease of use for nanospray and the design eliminates carryover effects
as the spray is produced directly from an orifice on each sample well of the chip.
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The
application of nanospray to screening DCLs will be of interest and could allow screens to
be carried out with sub-picomole quantities of target protein.
7.9 Advantages of FTMS for Screening Ligands for Protein Binding
A full discussion of the theory and performance of FTMS (Fourier transform mass spectro-
metry) is beyond the scope of this chapter; however, books and review articles are available
for the interested reader.
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For the purpose of this discussion, an outline of the basic prin-
ciples and performance characteristics of FTMS which are important for the measurement
