Protein and Metabolite Identification - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 243

is critical. In a bottom-up approach, all identi ed

phosphorylated peptides will be grouped into
a single protein sequence, making it impossible
to readily differentiate active and inactive popu-
lations. In top-down proteomics, the intact
proteins themselves are measured, allowing
eachmodi

proteins will precipitate out under conditions
used for reversed-phase chromatography.
Reversed-phase separations of intact proteins
do not exhibit the high resolution that peptides
do, nor do they give the same quality peak shape;
therefore, tools such as monolithic columns may
be necessary for top-down protein identi

ed variant to produce a unique signal.
Therefore, the various modi

cation.
Top-down proteomics has existed for several
years; Kelleher recently published a study that
illustrates the tremendous potential of this tech-
nology for characterizing proteomes. 7 A four-
dimensional

ed versions of the
protein can be accurately assigned.
The primary advantage of top-down MS is
that the molecular weight of the intact protein
is experimentally measured; therefore, the
contributions from each amino acid are recorded
in one signal. However, the mass of the intact
protein is insuf

separation system comprised

sequentially isoelectric focusing, gel-eluted

liquid fraction entrapment electrophoresis,

cient to identify an unknown
protein, even with the high mass measurement
accuracy instruments used today. The protein
must be fragmented similar to peptides analyzed
using MS 2 . For intact proteins, electron-capture
dissociation (ECD) and electron-transfer dissoci-
ation (ETD) are two methods that have demon-
strated excellent promise in fragmenting intact
proteins. Unfortunately, the sensitivity of identi-
fying intact proteins via top-down approaches is
not as great as bottom-up approaches for
peptides. Top-down fragmentation methods
produce a larger number of lower intensity frag-
ments than CID of peptides; therefore, one or
two orders of magnitude more material

nanocapillary liquid chromatography, and

finally MS was used to identify more than
3,093 proteins within human cells via top-
down proteomics. The identi ed proteins origi-

nated from 1,043 gene products, with the
proteins originating from the same gene having
been processing via different PTMs, RNA
splicing, and proteolysis events. Proteins greater
than 100,000 Da in molecular weight and
membrane proteins containing 11 transmem-
brane helices were identi

ed. Being able to iden-
tify more than 3,000 proteins brings top-down
proteomics into the same arena as bottom-up
methods and suggests that in the near future,
measuring intact proteins at this scale will
contribute to a fuller and more accurate blue-
print of the proteins encoded within the human
genome.

is

required for intact protein identi cation

compared to typical peptide identi cation.

Although development in MS technology is
critical for optimizing top-down protein identifi- -

cation, continuing improvements in sample
preparation steps are also necessary. Protein
solubility is very important. In bottom-up identi-

METABOLITE IDENTIFICATION IN
GLOBAL METABOLOMICS

fication, proteins are reduced to peptides that
overall are more soluble than intact proteins.
Because membrane proteins have much different
solubility requirements than those within the
cytosol, conditions must be established that will
solubilize as much as the proteome as possible.
In addition, although peptides are readily frac-
tionated using a variety of chromatographic
techniques, a signi

Mass spectrometry and nuclear magnetic
resonance (NMR) spectroscopy are the two
most widely used techniques for the analysis of
the metabolome. Global metabolomics is a study
that relies on the detection, identi cation, and

quantitation of as many metabolites as analyti-
cally possible in biological samples in search of
variations that can be used to discriminate

cant percentage of intact