Biology Reference
In-Depth Information
A proteome is the collection of proteins that are
present in a biological entity
patients in the US, the mass spectrometer does not have
the extendable throughput to manage 680 million
samples. In the future these analyses will be done by
microfluidic chips with ELISA (antibody) assays for
each protein. A recent microfluidic protein chip has
been designed with 50 ELISA assays for blood proteins
that can be analyzed from 200 nL of plasma in about 5
minutes across a dynamic range of close to 10
5
and
a sensitivity in the mid-atomole range
[39]
.Webelieve
that the potential for this chip can be extended to
thousands of protein measurements. Generating pairs of
antibodies for this many proteins would be extremely
expensive and time-consuming, and, moreover, anti-
bodies are not very stable. Accordingly, one will have to
develop more effective, stable and scalable protein-
capture agents. Aptamers (for example, 60-mers of
DNA) and trimeric or tetrameric peptide fragments (e.g.,
D
amino acid 6-mers) appear to be interesting candidates
as new protein-capture agents
[48,49]
.
The mass spectrometer is one of the most powerful
approaches for analyzing metabolites
[35,50]
.The
challenges for metabolite analysis are generally similar
to those of proteins (distinguishing the enormous
number of different metabolites, facilitating their
identification dealing with their broad dynamic ranges
of expression, etc.).
a cell, an organ, the
blood or an individual. The Human Genome Project has
given us the sequences of most of the human proteins
(and their tryptic peptides) and this has enable mass-
spectrometry-based proteomics. Mass spectrometry can
identify (and quantify) tryptic peptides of the proteins
(those generated by the proteolytic enzyme trypsin
cleaving either at the amino acid residues lysine or
arginine). The mass spectrometer has the ability to
separate and determine the mass to charge ratios of the
tryptic peptides (and thus identify them). Quantification
is achieved by determining the frequency of a particular
tryptic peptide in a proteome mixture and averaging the
frequencies of all the identifiable tryptic peptides shared
by a particular protein.
Mass spectrometry has been used in two ways. A
shotgun proteome analysis attempts to quantify all the
tryptic peptides present in the given proteome. This
procedure permits only the more dominant proteins to
be quantified accurately because of the limited dynamic
range and the fact that the peptides of rare proteins will
be seen rarely, if at all. A targeted proteome analysis
permits 100 proteins to be identified in a complex
mixture by synthesizing isotopically labeled 'standard'
peptides to be compared against their counterpart
peptides in the proteome mixture. The triple quadrapole
mass spectrometer has the ability to search out the
peptides from the proteins that will be quantified by
these assays. The targeted proteomic approach is called
selective or multiple reaction monitoring (SRM or
MRM) mass spectrometry. Recently, standard assays
have been developed for most of the 20 000 or so human
proteins
[47]
: just as the Human Genome Project
'democratized' all human genes by making them
accessible to every biologist, so this proteome project
has 'democratized' human proteins. Mass spectrometry
can also be used to analyze the proteins present in
organelles and can analyze those proteins interacting
with one another (after pulling down and purifying the
interacting protein complexes with specific antibodies).
Mass spectrometry has also been used to look at trans-
lational chemical modifications and the protein forms
arising from alternative RNA splicing.
The SRM assays have been used in assaying brain
and liver organ-specific blood proteins, both in the
organs and in the blood. As noted above in the section
discussing 'blood as a window', one would like to
contemplate the ability to analyze, say, 50 organ-specific
proteins from each of 50 different human organs, and to
follow for each patient's protein footprint across time to
assess health vs. disease. As one contemplates the
possibility of analyzing organ-specific blood proteins
several times a year in the blood of the 340 million
e
Single-cell analyses. Most of our understanding of
development, physiological responses and the initia-
tion and progression of disease comes from studies that
assess populations of cells. In the future the analyses of
large numbers of individual cells will become impor-
tant and standard practice
analyses that allow the
genomes, epigenomes, transcriptomes, miRNAomes,
proteomes, metabolomes, and interactomes within
single cells to be determined. Avariety of microfluidic
and nanotechnologic approaches are being applied to
these problems. Single-cell analyses will allow us to
answer two fundamental questions. First, do discrete,
quantized populations of cells exist within given
organs? Preliminary results suggest that the answer to
this question is yes. The fundamental issue is: what are
the biological roles of these quantized populations? For
example, they may represent a series of transition
intermediates on their waytoafinalendstage.Alter-
natively, they may represent discrete populations of
cells each with a separate function within the organ.
Second, the expression of some of the information
molecules within individual cells may behave in
a stochastic manner. Single-cell analyses, properly
executed, will permit us to distinguish between quan-
tized cell populations and stochastic variability. We
believe that single-cell analyses will be a critical tool in
the future for deciphering biological complexity.
e
