Instrumentation for LC-MS/MS in Proteomics - LC-MS/MS in Proteomics: Methods and Applications - page 47

2. Liquid
Chromatography
Instrumentation

Although electrospray starts from the solution phase, the mass
spectrometer is ultimately detecting gaseous ions. Hence, dur-
ing the ionization process, all the solvent has to be removed.
The process of ESI converts a solution into a mist of charged
droplets. These droplets shrink as the solvent is evaporated and
when the charge density in the droplet reaches a critical level,
coulombic repulsion causes desorption of charged gaseous ions
from the droplet ( 3 , 4 ) . This process continues; as solvent contin-
ues to evaporate, droplets get smaller and more ions are ejected
into the gaseous phase. Unsurprisingly, this conversion of liquid
sample into gaseous ions is more efficient when less solvent is
present. Hence, electrospray is referred to as a concentration-
sensitive process. This means that the smaller the volume of sam-
ple introduced, the better the efficiency and sensitivity of the pro-
cess. Increasing sample concentration can be achieved in two ways
using liquid chromatography. First, use of a narrower column and
lower flow rate will cause elution in smaller volumes. Second, by
improving the resolution of separation, the same amount of sam-
ple will elute in a narrower profile, giving a higher concentration
at the maxima of peak elution.
Nanospray is more sensitive than ESI approaches at higher
flow rates ( 5 ) ; so sub-microliter flow rates are typically used
for proteomic analyses. Many chromatographic systems cannot
natively produce reliable gradients at these low flow rates due to
the presence of solvent-mixing chambers in the plumbing that
have too large volumes in comparison to the solvent flow rate,
leading to inconsistent solvent mixing and irreproducible gradi-
ents. Hence, pressure-based flow-splitting systems are commonly
employed and built into the chromatography system. Generally, a
split in the range of 1:100 to 1:1,000 is used post-pump but prior
to the separation column, allowing efficient mixing of solvents
prior to chromatography to produce consistent separations, albeit
with large amounts of solvent waste. Recently, some systems have
been developed that use air pressure as the pumping mechanism
and allow splitless delivery of reproducible nanoliter per minute
flow rates.
Different stationary phases in chromatography columns pro-
vide variable levels of resolution. Reverse-phase chromatography
is highly compatible with subsequent mass spectrometric anal-
ysis due to the lack of salts in the buffers and provides rel-
atively high-resolution separation, so is the dominant separa-
tion method in use for proteomic analysis. Most reverse-phase
stationary phases for LC-MS analysis consist of silica beads of
3-5 μ m in diameter with alkyl chains of either eight or eighteen