Essential Cell Biology 5th edition

How Proteins Are Studied
161
Figure 4−56 Mass spectrometry can be used to identify proteins
by determining the precise masses of peptides derived from them.
As indicated, this in turn allows proteins of interest to be produced in
the large amounts needed for determining their three-dimensional
structure. In this example, a protein of interest is excised from a
polyacrylamide gel after two-dimensional electrophoresis (see Panel
4−5, p. 167) and then digested with trypsin. The peptide fragments
are loaded into the mass spectrometer, and their exact masses are
measured. Genome sequence databases are then searched to find the
protein encoded by the organism in question whose profile matches
this peptide fingerprint. Mixtures of proteins can also be analyzed in
this way. (Image courtesy of Patrick O’Farrell.)
mixtures are frequently analyzed using tandem mass spectrometry. In this
case, after the peptides pass through the first mass spectrometer, they
are broken into even smaller fragments and analyzed by a second mass
spectrometer.
Although all the information required for a polypeptide chain to fold is
contained in its amino acid sequence, only in special cases can we reliably
predict a protein’s detailed three-dimensional conformation—the
spatial arrangement of its atoms—from its sequence alone. Today, the
predominant way to discover the precise folding pattern of any protein
is by experiment, using x-ray crystallography, nuclear magnetic
resonance (NMR) spectroscopy, or most recently cryoelectron
microscopy (cryo-EM), as described in Panel 4–6 (pp. 168–169).
Genetic Engineering Techniques Permit the Large-Scale
Production, Design, and Analysis of Almost Any Protein
Advances in genetic engineering techniques now permit the production
of large quantities of almost any desired protein. In addition to making
life much easier for biochemists interested in purifying specific proteins,
this ability to churn out huge quantities of a protein has given rise to an
entire biotechnology industry (Figure 4−57). Bacteria, yeast, and cultured
mammalian cells are now used to mass-produce a variety of therapeutic
proteins, such as insulin, human growth hormone, and even the fertilityenhancing
drugs used to boost egg production in women undergoing in
vitro fertilization treatment. Preparing these proteins previously required
the collection and processing of vast amounts of tissue and other biological
products—including, in the case of the fertility drugs, the urine of
postmenopausal nuns.
The same sorts of genetic engineering techniques can also be employed
to produce new proteins and enzymes that contain novel structures or
perform unusual tasks: metabolizing toxic wastes or synthesizing lifesaving
drugs, for example. Most synthetic catalysts are nowhere near as
effective as naturally occurring enzymes in terms of their ability to speed
abundance
single protein spot excised from gel
N
PROTEINS PREDICTED FROM GENOME
SEQUENCES ARE SEARCHED FOR MATCHES
WITH THEORETICAL MASSES CALCULATED
FOR ALL TRYPSIN-RELEASED PEPTIDES
IDENTIFICATION OF PROTEIN
SUBSEQUENTLY ALLOWS ISOLATION
OF CORRESPONDING GENE
C
PEPTIDES PRODUCED
BY TRYPTIC DIGESTION
HAVE THEIR MASSES
MEASURED USING A
MASS SPECTROMETER
0 m
1600
z
(mass-to-charge ratio)
THE GENE SEQUENCE ALLOWS LARGE
AMOUNTS OF THE PROTEIN TO BE OBTAINED
BY GENETIC ENGINEERING TECHNIQUES
ECB5 04.56
Figure 4−57 Biotechnology companies
produce mass quantities of useful
proteins. Shown in this photograph are the
large, turnkey microbial fermenters used
to produce a whooping cough vaccine.
(Courtesy of Pierre Guerin Technologies.)