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MS analysis of complex peptide mixtures can be improved by
combinations of orthogonal chromatographic phases (i.e., based on
complementary selectivity for peptides) in which each fraction from
the upstream column is further separated on the downstream column
[26,34,35]. The most common arrangement includes separation by ion
exchange on the upstream column followed by analysis of each fraction
in the reversed-phase downstream (analytical) column. Online integration
of multidimensional chromatography and tandem mass spectrometry
(figure 1.5) provides a robust automated platform for global proteome
analysis by shotgun sequencing [27,28,33]. Together with the Sequest
database searching algorithm, online multidimensional chromatogra-
phy-tandem mass spectrometry forms the core of multidimensional
protein identification technology (MudPIT) platform (figure 1.5).
Shotgun sequencing has been performed using one-dimensional
chromatography together with high-accuracy measurement of mass
(by FTICR) to assign accurate mass tags for peptides [36]. In subsequent
analyses, accurate mass tag and retention times during 1D chromato-
graphy of the peptide form a unique identifier which can then be used
for large-scale proteomic experiments [37].
More narrowly focused proteomic approaches are being used to
monitor subpopulations of peptides with distinct physicochemical
properties. Most of these approaches use a number of affinity methods
in a preparative rather than online analytical fashion. The most notable
examples are phosphopeptides enriched by immobilized metal affinity
chromatography (IMAC) and glycosylated peptides isolated by lectin
chromatography. Amino acid specific selection has been demonstrated
by diagonal chromatography where a sample is run twice: before and
after a chemical reaction specific to an amino acid of interest [38].
However, most of these methods focus on certain classes of peptides
while discarding the rest of the sample. Some combination of affinity
techniques with multidimensional separations will eventually provide
an integrated analysis of specific modified peptides in the context of
their parent proteins.
LC techniques offer a wide variety of choices for large-scale peptide
analysis, but only few tailored methods for specific classes of lipids. Gross
and colleagues introduced intrasource separation by using the electrical
properties of lipids. In this method, the polarity of ESI (positive/negative
ion) has been used to separately analyze classes of lipids that may ionize
preferentially or with stronger signals as positive ions (neutral polar
species) or negative ions (anionic species) [10,23]. 2D ESI has also been
applied to multidimensional analysis of phosphopeptides, where the
negative ion polarity is used for improved detection of phosphopeptides
that are further sequenced by MS/MS in positive ion polarity [39].
Capillary electrophoresis (CE) separates bioanalytes based on size-
to-charge ratio and constitutes yet one more possible dimension of
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