Proteomics: Applications and Advances 18.3 Proteomics and Marine Biotechnology 477
Part C | 18.4
label the proteins with the fluorescent CyDyes, ranging from 1 to < 0:1 ng protein per spot with minimal
or saturation labeling, respectively [18.8]. Once differentially expressed protein spots have been identified,
either manual spot picking or a robotic spot picker is
used to obtain samples of the spots for identification
by MS.
MS is widely used for the identification and quantitation of proteins in biological samples [18.9, 10].
The majority of protocols involve trypsin digestion
of the protein in each of the sampled spots to generate peptides with a mass range that is more conducive to MS, since they can be effectively ionized
by electrospray [18.4, 9]. The resulting peptide mixture is fractionated by reverse phase liquid chromatography and analyzed by tandem mass spectrometry
(MS/MS) [18.10]. Briefly, MS/MS involves ionization
of the peptides followed by separation of the peptide
ions according to their mass to charge ratio and further
fragmentation for sequence analysis [18.2].
Data obtained by MS/MS includes information concerning the peptide masses, fragment ion masses, and
intensities, which is transformed into a peak list that
is searched against a protein database of in silicodigested proteins using open-source or proprietary software packages [18.2]. If insufficient sequence data is
available regarding the organism under investigation, de
novo peptide sequences can be used to interrogate protein sequence databases. Besides identification of the
proteins present in a particular biological sample, MS
can also be used to quantitate the proteins present in
individual samples. Isobaric tags for relative and absolute quantification (iTRAQ) is a widely used chemical
labeling technique that allows quantitative comparison
of protein samples from up to eight treatments simultaneously [18.11, 12]. Since the eight isobaric mass
tags result in different mass spectra following peptide
fragmentation, iTRAQ allows simultaneous identification and quantitation of the peptides in the eight
samples [18.2].
18.3 Proteomics and Marine Biotechnology
The marine environment covers 70% of the Earth’s
surface and is inhabited by a broad diversity of organisms [18.13, 14]. The latter reflects the fact that life
originated in the sea approximately 3:8 million years
ago and has adapted to the wide range of habitats that
comprise the marine environment, many of which are
considered extreme [18.14].
Given the extent of the marine environment and the
biodiversity it contains, marine biotechnology promises
to deliver a vast array of commercially important products that include novel bioactive compounds, enzymes,
and biopolymers. In addition, technology has been
developed that has allowed aquaculture of a number of fish and shellfish species to provide an increasingly important source of protein for human
consumption.
Indeed, marine aquaculture production is increasing steadily, achieving a 20% growth in production
from 2004 (16:7 mt) to 2009 (20:1 mt), while wild fisheries harvest decreased by almost 5% from 83:8 to
79:9 mt over the same period [18.15]. Despite the many
potential benefits that may be derived from marine organisms, the health of the marine environment is being
damaged enormously due to rapidly elevating levels of
anthropogenic pollution, over-fishing, and the effects
of climate change. In fact, a significant proportion of
the world’s population lives within 75 mi of an ocean
coast and the number is expected to continue to increase together with a substantial impact on the marine
environment [18.16].
In order for us to derive the benefits that the marine
environment offers humanity through marine biotechnology and mitigate the damage done to the marine
environment, it is necessary for us to utilize modern technologies to better understand the functioning
of the organisms that inhabit the marine environment
in terms of their ecology and biology. As discussed
earlier, the omics era is generating new insights into
the molecular biology of living organisms at a rate
never before achieved. Proteomics provides a unique
opportunity to characterize the phenotype of marine
organisms, which dynamically responds to changing
environmental conditions and associated stresses. Indeed, proteomics has a range of applications in marine
biotechnology, such as identification of unique bioactive gene products, characterization of the function
and regulation of metabolic processes, determination
of biodiversity, and identification of biomarkers of
pollution and disease. The following sections of this
chapter will discuss many areas of marine biotechnology which have been investigated using a proteomic
approach.
Part C | 18.4
label the proteins with the fluorescent CyDyes, ranging from 1 to < 0:1 ng protein per spot with minimal
or saturation labeling, respectively [18.8]. Once differentially expressed protein spots have been identified,
either manual spot picking or a robotic spot picker is
used to obtain samples of the spots for identification
by MS.
MS is widely used for the identification and quantitation of proteins in biological samples [18.9, 10].
The majority of protocols involve trypsin digestion
of the protein in each of the sampled spots to generate peptides with a mass range that is more conducive to MS, since they can be effectively ionized
by electrospray [18.4, 9]. The resulting peptide mixture is fractionated by reverse phase liquid chromatography and analyzed by tandem mass spectrometry
(MS/MS) [18.10]. Briefly, MS/MS involves ionization
of the peptides followed by separation of the peptide
ions according to their mass to charge ratio and further
fragmentation for sequence analysis [18.2].
Data obtained by MS/MS includes information concerning the peptide masses, fragment ion masses, and
intensities, which is transformed into a peak list that
is searched against a protein database of in silicodigested proteins using open-source or proprietary software packages [18.2]. If insufficient sequence data is
available regarding the organism under investigation, de
novo peptide sequences can be used to interrogate protein sequence databases. Besides identification of the
proteins present in a particular biological sample, MS
can also be used to quantitate the proteins present in
individual samples. Isobaric tags for relative and absolute quantification (iTRAQ) is a widely used chemical
labeling technique that allows quantitative comparison
of protein samples from up to eight treatments simultaneously [18.11, 12]. Since the eight isobaric mass
tags result in different mass spectra following peptide
fragmentation, iTRAQ allows simultaneous identification and quantitation of the peptides in the eight
samples [18.2].
18.3 Proteomics and Marine Biotechnology
The marine environment covers 70% of the Earth’s
surface and is inhabited by a broad diversity of organisms [18.13, 14]. The latter reflects the fact that life
originated in the sea approximately 3:8 million years
ago and has adapted to the wide range of habitats that
comprise the marine environment, many of which are
considered extreme [18.14].
Given the extent of the marine environment and the
biodiversity it contains, marine biotechnology promises
to deliver a vast array of commercially important products that include novel bioactive compounds, enzymes,
and biopolymers. In addition, technology has been
developed that has allowed aquaculture of a number of fish and shellfish species to provide an increasingly important source of protein for human
consumption.
Indeed, marine aquaculture production is increasing steadily, achieving a 20% growth in production
from 2004 (16:7 mt) to 2009 (20:1 mt), while wild fisheries harvest decreased by almost 5% from 83:8 to
79:9 mt over the same period [18.15]. Despite the many
potential benefits that may be derived from marine organisms, the health of the marine environment is being
damaged enormously due to rapidly elevating levels of
anthropogenic pollution, over-fishing, and the effects
of climate change. In fact, a significant proportion of
the world’s population lives within 75 mi of an ocean
coast and the number is expected to continue to increase together with a substantial impact on the marine
environment [18.16].
In order for us to derive the benefits that the marine
environment offers humanity through marine biotechnology and mitigate the damage done to the marine
environment, it is necessary for us to utilize modern technologies to better understand the functioning
of the organisms that inhabit the marine environment
in terms of their ecology and biology. As discussed
earlier, the omics era is generating new insights into
the molecular biology of living organisms at a rate
never before achieved. Proteomics provides a unique
opportunity to characterize the phenotype of marine
organisms, which dynamically responds to changing
environmental conditions and associated stresses. Indeed, proteomics has a range of applications in marine
biotechnology, such as identification of unique bioactive gene products, characterization of the function
and regulation of metabolic processes, determination
of biodiversity, and identification of biomarkers of
pollution and disease. The following sections of this
chapter will discuss many areas of marine biotechnology which have been investigated using a proteomic
approach.