The Proteomics Ensemble—What is Entailed in Proteomics
This section gives an overview of the whole process of protein
profiling, from sample selection and isolation to data
evaluation and analysis. Proteomics combines know-how
from biology, engineering, chemistry, bioinformatics, and other
fields to simultaneously understand the function and interaction
of multiple constituents of a proteome. By its very nature, this
Herculean task is complex and a complete analysis of the
entire process is beyond the means of this report. However,
this section serves to highlight the flow of sample and
information though the proteomics process. The aim of this
section is to understand the dependency of each individual
module to others up- and down-stream of it. Such techniques
as 2-D gel electrophoresis and mass spectrometry which are
integral to the proteomics field will be reviewed in more detail
in later sections. The more simple, traditional, and peripheral
tools used to bridge sample selection at the onset and data
analysis at the conclusion will be discussed in this section
briefly. The big picture helps to highlight the competencies and
shortcomings of the current technology. For example,
bottlenecks in the process are revealed. These bottlenecks
can be both in terms of speed of handling or in terms of
current shortcomings in certain assays or tools.
Proteomics and the study of proteins is inherently more
complex than genomics, the study of DNA and the genome.
DNA is made of four nucleotide of generally similar structure;
therefore DNA can be separated based on its molecular
weight very easily. DNA detection and replication is very
routine through techniques such as polymerase chain reaction
(PCR), and DNA sequencing is simple, inexpensive, and
rapid. DNA is very stable under most laboratory conditions,
also.
Proteins are made of twenty different amino acids that are
very diverse in size, charge, hydrophobicity, and structure.
Since proteins are encoded by DNA and translated from
messenger RNA, proteins cannot be replicated directly in the
laboratory and detection often requires indirect assays with
various stains and dyes. Although chemical sequencing of
proteins predates all DNA sequencing protocols, protein
sequencing is still a complex procedure. Recent advances
have yielded more rapid and high fidelity protocols for
sequencing of peptides, but sequencing of proteins (not merely
peptides of a few amino acids) still demands specialized
expertise and instrumentation. Protein sequencing is more
expensive and time consuming, also. Furthermore, the mere
detection and sequencing of proteins is often inadequate for
understanding their biological role. Proteins are modified
post-translationally by multiple processes that can be both
irreversible and transient. These modifications can play an
important role in the appropriate function of each protein.
Additionally, proteins often require other proteins to reveal
their true and complete function. Hence, it is rarely possible to
study one protein in itself. The interaction of proteins with
each other and/or sub cellular organelles can be very
instrumental in the well being of the cell and the organism.
Considering the aforementioned differences between
proteomic and genomic studies, we must recall the questions
that proteomics seeks to answer before delineating a path
between those questions and the answers (sample to data).
And finally, we must understand what each step in the
pathway demands in terms of resources and information.
Proteomics aims to directly study the role and function of
proteins in tissues and cells. The ultimate goal is to study the
interaction of multiple proteins in healthy, diseased, and
experimental conditions. Such global studies will enable the
investigator to understand the holistic effects of a particular
therapeutic agent or experimental intervention. To this end,
proteomics requires the ability to separate and isolate all the
constituents of a proteome. These pure or near homogeneous
protein isolates must be detectable and in a form conducive to
analysis.
All proteomics studies fall into the following [three] general
categories: differential protein display (including changes in
quantity), protein characterization (including post-translational
modifications), and protein-protein interaction (including
activity assays). All of these demand careful isolation of tissues
or cells and proper sample preparation at the onset of the
study. The correct choice for starting material, and proper
preparation of it, can dictate the success of the whole
procedure, because most studies rely on either purity of the
sample or valid comparisons (untainted) between two
samples.
The very first step is to choose and catalogue the proper
crude samples. In basic research, the investigator has great
discretion over this manner. For example, in differential
protein display between two in vitro tissue culture samples, the
treated and untreated cells are easily distinguishable. The
experimental and control samples can be chosen based on
their identical, or at least, similar pedigree to minimize spurious
and irrelevant differences between the two. The only
distinctions, at least theoretically, in the protein profile of the
two samples will be due to the known intervention or
therapeutic. Therefore, the selection of starting material in such
a scenario is simple. However, this is not the case for most
applications, even in basic science. The target cells are often
intermixed with a variety of other cell types or unafflicted cells
of the same kind. All proteomics protocols demand the
separation of the cells of interest from the rest of the tissue.
Otherwise, differential protein display, protein
characterization, and protein-protein interaction studies will be
tainted with the constituents of the background tissue. |
