Mass Spectrometry in Systems Biology - Systems Biology: Networks, Models, and Applications - page 13

for complete structural characterization. Identification by comparison
to a MS/MS spectrum of a pure synthetic analog is also applied to
metabolites analysis because of their considerable chemical diversity [24].
Elucidation of bioanalyte structure to fine details, particularly when
comparing differences in structure, might require more than MS/MS
fragmentation. Recent developments in mass spectrometry instrumen-
tation have introduced routine analysis of bioanalytes structures by
multiple stages (e.g., MS 3 and higher) of fragmentation in combination
with neutral loss detection in a data-dependent manner (see Mass spec-
trometers section above).

Separation of Bioanalytes
Separation of bioanalytes prior to presentation to mass spectrometry
analysis is a key element for global analysis of biomolecules regardless
of their nature: proteins, peptides, nucleic acids, lipids, carbohydrates,
pharmaceuticals, organic molecules, and so on. Efficient separation is
required before presenting biomolecules to the mass spectrometer.
Application of MS to biological molecules was facilitated by the develop-
ment of separation techniques, particularly of capillary chromatography
interfaced online with ESI that reduced the flow rate of analysis and
increased its sensitivity. High-resolution separations of complex biolog-
ical mixtures are important to decrease ion suppression during ionization
and to increase dynamic range. For peptides, capillary liquid chro-
matography (LC) simplifies complicated mixtures before they reach
the ionization source. Alternatively, for lower molecular weight com-
pounds (i.e., metabolites), capillary gas chromatography (GC) separates
complex mixtures if bioanalytes are volatile or can be derivatized to
volatile compounds. GC coupled online with MS (GC-MS) is the main-
stay of metabolomics analysis. In this section, we will emphasize LC
applications to large-scale analysis of peptides for proteomics.
Proteomics is a biological assay [25], where the identification of pro-
teins is made either by reconstituting sequence information from
shotgun analysis of peptides (bottom-up approach) [26-28] or by direct
molecular weight measurements and subsequent fragmentation of pro-
teins (top-down approach) [29-31].
In bottom-up proteomics, proteolytically digested proteins (either
in solution or in gel) are sequenced by tandem mass spectrometry.
Samples are loaded on capillary columns interfaced online with a mass
spectrometer. A pressure vessel is used to load volumes of up to 1 ml
in the capillary columns. Microcolumn/capillary chromatography is
the workhorse of current research in proteomics (the inner diameter of
a capillary column is 75-100

m) [32]. The reader is referred to ref. [33]
for a comprehensive tutorial on building and assembling nanoflow-
HPLC columns and micro-ESI as well as for sample loading on capillary
columns.

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