Biology Reference
In-Depth Information
Quantitative proteomics experiments produce input data for sys-
tems biology. From this prospective, protein expression levels should
be statistically accounted for at less than two-fold difference in order to
be useful in the description of a complex system [58]. Recently, a suite
of methods using MudPIT as core technology has been developed to
accurately quantify small differences in protein expression levels en
masse, by using metabolically labeled organisms [45,59] and sophisti-
cated software for quantitative proteomics [59,60].
Global quantitation of cellular lipids employs a different strategy as
compared to stable isotope labeling. Because ESI efficiency depends
mostly on lipid polar head, classes of lipids can be quantified by a single
internal standard [23]. This represents a practical advantage as it would
be close to impossible to synthesize stable-isotope internal standards for
thousands of lipids. In spite of recent advances in lipidomics, methodo-
logical developments have to mature in widespread technologies.
However, as we will see in the next section, proteomics technologies
already fully benefit from the individual analytical aspects of both LC
and MS as well as their use in concerted manner as LC-MS.
Platforms for Proteomics
Online assembly of LC and MS constitutes a continuum that generates
multidimensional data: chromatospectrograms. Chromatospectrograms
are the usual chromatographic profiles that embed mass spectral infor-
mation, MS and/or MS/MS spectra. Combinations of chromatographic
profiles and m / z intensities are used for extracting quantitative infor-
mation without using stable isotope labeling [61].
Three parameters completely describe a biomolecule in an LC-MS
experiment: a series of m / z values (present in MS and MS/MS spectra),
its retention time (RT), and its corresponding elution profile (i.e., the
area under the curve that is proportional with the amount). The data
generated from an experiment can be re-inspected using any of these
parameters and information about its value creates the opportunity for
hypothesis-driven mass spectrometry. For example, a comparison of
two states can highlight only those ions showing a change in abundance
and those ions can then be analyzed using a tandem mass spectrome-
try experiment to determine the identity of the molecule. Instruments
used for this strategy combine MALDI with quadrupole ion traps or
Q-TOF detection. By its nature, MALDI would allow for an analysis to
be repeated until the sample is consumed.
In hypothesis-driven mass spectrometry there are two stages of
analysis: (a) survey of the sample in high-throughput manner to gener-
ate a hypothesis (usually by MALDI-MS or MS/MS) followed by (b)
testing of the hypothesis with a different strategy (that could either use
MALDI MS n or LC-MS/MS). MALDI quadrupole ion trap allows for
in-depth interrogation of proteomic samples based on the prediction of
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