2

Bioinformatics Applied to Proteomics

Simone Cristoni1 and Silvia Mazzuca2
1Ion Source Biotechnologies srl, Milano,
2Plant Cell Physiology laboratory, Università della Calabria, Rende,
Italy

1. Introduction
Proteomics is a fundamental science in which many sciences in the world are directing their
efforts. The proteins play a key role in the biological function and their studies make
possible to understand the mechanisms that occur in many biological events (human or
animal diseases, factor that influence plant and bacterial grown). Due to the complexity of
the investigation approach that involve various technologies, a high amount of data are
produced. In fact, proteomics has known a strong evolution and now we are in a phase of
unparalleled growth that is reflected by the amount of data generated from each
experiment. That approach has provided, for the first time, unprecedented opportunities to
address biology of humans, animals, plants as well as micro-organisms at system level.
Bioinformatics applied to proteomics offered the management, data elaboration and
integration of these huge amount of data. It is with this philosophy that this chapter was
born.
Thus, the role of bioinformatics is fundamental in order to reduce the analysis time and to
provide statistically significant results. To process data efficiently, new software packages
and algorithms are continuously being developed to improve protein identification,
characterization and quantification in terms of high-throughput and statistical accuracy.
However, many limitations exist concerning bioinformatic spectral data elaboration. In
particular, for the analysis of plant proteins extensive data elaboration is necessary due to
the lack of structural information in the proteomic and genomic public databases. The main
focus of this chapter is to describe in detail the status of bioinformatics applied to proteomic
studies. Moreover, the elaboration strategies and algorithms that have been adopted to
overcome the well known limitations of the protein analysis without database structural
information are described and disclosed.
This chapter will get rid of light on recent developments in bioinformatic and data-mining
approaches, and their limitations when applied to proteomic data sets, in order to reinforce
the interdependence between proteomic technologies and bioinformatics tools. Proteomic
studies involve the identification as well as qualitative and quantitative comparison of
proteins expressed under different conditions, together with description of their properties
and functions, usually in a large-scale, high-throughput format. The high dimensionality of
data generated from these studies will require the development of improved bioinformatics
tools and data-mining approaches for efficient and accurate data analysis of various
26 Systems and Computational Biology – Bioinformatics and Computational Modeling

biological systems (for reviews see, Li et al, 2009; Matthiesen & Jensen, 2008; Wright et al,
2009). After a rapid moving on the wide theme of the genomic and proteomic sciences, in
which bioinformatics find their wider applications for the studies of biological systems, the
chapter will focus on mass spectrometry that has become the prominent analytical method for
the study of proteins and proteomes in post-genome era. The high volumes of complex spectra
and data generated from such experiments represent new challenges for the field of
bioinformatics. The past decade has seen an explosion of informatics tools targeted towards
the processing, analysis, storage, and integration of mass spectrometry based proteomic data.
In this chapter, some of the more recent developments in proteome informatics will be
discussed. This includes new tools for predicting the properties of proteins and peptides
which can be exploited in experimental proteomic design, and tools for the identification of
peptides and proteins from their mass spectra. Similarly, informatics approaches are required
for the move towards quantitative proteomics which are also briefly discussed. Finally, the
growing number of proteomic data repositories and emerging data standards developed for
the field are highlighted. These tools and technologies point the way towards the next phase of
experimental proteomic and informatics challenges that the proteomics community will face.
The majority of the chapter is devoted to the description of bioinformatics technologies
(hardware and data management and applications) with particular emphasis on the
bioinformatics improvements that have made possible to obtain significant results in the
study of proteomics. Particular attention is focused on the emerging statistic semantic,
network learning technologies and data sharing that is the essential core of system biology
data elaboration.
Finally, many examples of bioinformatics applied to biological systems are distributed along
the different section of the chapter so to lead the reader to completely fill and understand
the benefits of bioinformatics applied to system biology.

2. Genomics versus proteomics

There have been two major diversification paths appeared in the development of
bioinformatics in terms of project concepts and organization, the -omics and the bio-. These
two historically reflect the general trend of modern biology. One is to go into molecular
level resolution. As one of the -omics and bio- proponents, the -omics trend is one of the
most important conceptual revolutions in science. Genetic, microbiology, mycology and
agriculture became effectively molecular biology since 1970s. At the same time, these fields
are now absorbing omics approach to understand their problems more as complex systems.
Omics is a general term for a broad discipline of science and engineering for analyzing the
interactions of biological information objects in various omes. These include genome,
proteome, metabolome, expressome, and interactome. The main focus is on mapping
information objects such as genes, proteins, and ligands finding interaction relationships
among the objects, engineering the networks and objects to understand and manipulate the
regulatory mechanisms and integrating various omes and omics subfields.
This was often done by researchers who have taken up the large scale data analysis and
holistic way of solving bio-problems. However, the flood of such -omics trends did not
occur until late 1990s. Until that time, it was by a relatively small number of informatics
advanced people in Europe and the USA. They included Medical Research Council [MRC]
Cambridge, Sanger centre, European Bioinformatics Institute [EBI], European Molecular
Bioinformatics Applied to Proteomics 27

Biology Laboratory [EMBL], Harvard, Stanford and others. We could clearly see some
people took up the underlying idea of -ome(s) and -omics quickly, as biology was heading
for a more holistic approach in understanding the mechanism of life. Whether the suffix is
linguistically correct or not, the -omics suffix changed in the way many biologists view their
research activity. The most profound one is that biologists became freshly aware of the fact
that biology is an information science more than they have thought before.
In general terms, genomics is the -omics science that deals with the discovery and noting of
all the sequences in the entire genome of a particular organism. The genome can be defined
as the complete set of genes inside a cell. Genomics, is, therefore, the study of the genetic
make-up of organisms. Determining the genomic sequence, however, is only the beginning
of genomics. Once this is done, the genomic sequence is used to study the function of the
numerous genes (functional genomics), to compare the genes in one organism with those of
another (comparative genomics), or to generate the 3-D structure of one or more proteins
from each protein family, thus offering clues to their function (structural genomics). At
today a list of sequenced eukaryotic genomes contains all the eukaryotes known to have
publicly available complete nuclear and organelle genome sequences that have been
assembled, annotated and published. Starting from the first eukaryote organism
Saccharomyces cerevisiae to have its genome completely sequenced at 1998, further genomes
from 131 eukaryotic organisms were released at today. Among them 33 are Protists, 16 are
Higher plants, 26 are Fungi, 17 are Mammals Humans included, 9 are non-mammal animals
,10 are Insects, 4 Nematodes, remaining 11 genomes are from other animals and as we write
this chapter, others are still to be sequenced and will be published during the editing of this
book. A special note should be paid to the efforts of several research teams around the
world for the sequencing of more than 284 different Eubacteria, whose numbers increased
by 2-3% if we consider the sequencing of different strains for a single species; also a list of
sequenced archaeal genomes contains 28 Archeobacteria known to have available complete
genome sequences that have been assembled, annotated and deposited in public databases.
A striking example of the power of this kind of -omics and knowledge that it reveals is that
the full sequencing of the human genome has dramatically accelerated biomedical research
and diagnosis forecast; very recently Eric S. Lander (2011) explored its impact, in the decade
since its publication, on our understanding of the biological functions encoded in the human
genome, on the biological basis of inherited diseases and cancer, and on the evolution and
history of the human species; also he foresaw the road ahead in fulfilling the promise of
genomics for medicine.
In the other side of living kingdoms, genomics and biotechnology are also the modern tools
for understanding plant behavior at the various biological and environmental levels. In The
Arabidopsis Information Resource [TAIR] a continuously updated database of genetic and
molecular biology data for the model higher plant Arabidopsis thaliana is maintained (TAIR
Database, 2009)
This data available from TAIR include the complete genome sequence along with gene
structure, gene product information, metabolism, gene expression, DNA and seed stocks,
genome maps, genetic and physical markers, publications, and information about the
Arabidopsis research community. Gene product function data is updated every two weeks
from the latest published research literature and community data submissions. Gene
structures are updated 1-2 times per year using computational and manual methods as well
as community submissions of new and updated genes.
28 Systems and Computational Biology – Bioinformatics and Computational Modeling

Genomics provides also boosting to classical plant breeding techniques, well summarized in
the Plants for the Future technology platform (http://www.epsoweb.eu/
catalog/tp/tpcom_home.htm). A selection of novel technologies come out that are now
permitting researchers to identify the genetic background of crop improvement, explicitly
the genes that contribute to the improved productivity and quality of modern crop varieties.
The genetic modification (GM) of plants is not the only technology in the toolbox of modern
plant biotechnologies. Application of these technologies will substantially improve plant
breeding, farming and food processing. In particular, the new technologies will enhance the
ability to improve crops further and, not only will make them more traceable, but also will
enable different varieties to exist side by side, enhancing the consumer’s freedom to choose
between conventional, organic and GM food. In these contexts agronomical important genes
may be identified and targeted to produce more nourishing and safe food; proteomics can
provide information on the expression of transgenic proteins and their interactions within
the cellular metabolism that affects the quality, healthy and safety of food. Taking
advantage of the genetic diversity of plants will not only give consumers a wider choice of
food, but it will also expand the range of plant derived products, including novel forms of
pharmaceuticals, biodegradable plastics, bio-energy, paper, and more. In this view, plant
genomics and biotechnology could potentially transform agriculture into a more
knowledge-based business to address a number of socio-economic challenges.
In systems biology (evolutionary and/or functionally) a central challenge of genomics is to
identify genes underlying important traits and describe the fitness consequences of variation
at these loci (Stinchcombe et al., 2008). We do not intend to give a comprehensive overview
of all available methods and technical advances potentially useful for identifying functional
DNA polymorphisms, but rather we explore briefly some of promising recent developments
of genomic tools from which proteomics taken its rise during the last twenty years,
applicable also to non model organisms.
The genome scan, became one of the most promising molecular genetics (Oetjen et al., 2010).
Genome scans use a large number of molecular markers coupled with statistical tests in
order to identify genetic loci influenced by selection (Stinchombe & Hoekstra, 2008). This
approach is based on the concept of ‘genetic hitch-hiking’ (Maynard Smith & Haigh, 1974)
that predicts that when neutral molecular markers are physically linked to functionally
important and polymorphic genes, divergent selection acting on such genes also affects the
flanking neutral variation. By genotyping large numbers of markers in sets of individuals
taken from one or more populations or species, it is possible to identify genomic regions or
‘outlier loci’ that exhibit patterns of variation that deviate from the rest of the genome due to
the effects of selection or treats (Vasemägi & Primmer 2005). An efficient way of increasing
the reliability of genome scans, which does not depend on the information of the genomic
location of the markers, is to exploit polymorphisms tightly linked to the coding sequences,
such as expressed sequence tag (EST) linked microsatellites (Vigouroux et al., 2002;
Vasemägi et al., 2005). Because simple repeat sequences can serve as promoter binding sites,
some microsatellite polymorphisms directly upstream of genes may have a direct functional
significance (Li et al., 2004).
EST libraries represent sequence collections of all mRNA (converted into complementary or
cDNA) that is transcribed at a given point in time in a specific tissue (Bouck & Vision, 2007).
EST libraries have been constructed and are currently being analyzed for many species
whose genomes are not completed. EST library also provide the sequence data for
Bioinformatics Applied to Proteomics 29

expression analysis using Quantitative real-time PCR (QPCR), as well as for transcription
profiling using microarrays and, finally, the EST database can be a valuable tool for
identifying new candidate polymorphism in proteins of specific interest. QPCR is a method
that can measure the abundance of mRNA (converted in cDNA) of specific genes (Heid et
al., 1996). The expression of a target gene can be related to the total RNA input, or it can be
quantified in relation to the expression of a reference gene, the housekeeping gene (HKG,
i.e. gene always expressed at the same level). Unfortunately, a universal reference gene that
is expressed uniformly, in all biological conditions in all tissues, does not exist. For each
experimental setup using QPCR, the choice of HKG must reflect the tissue used and the
experimental treatment.
While QPCR can only handle a few candidate genes, microrrays technology quantifies the
expression level of hundreds to thousands of genes simultaneously, providing a powerful
approach for the analysis of global transcriptional response (Yauk & Berndt, 2007). For
example, the analysis of mRNA via genomic arrays is one approach to finding the genes
differentially expressed across two kind of tissue or sample obtained under two
experimental conditions or to finding the genes that matter to organisms undergoing
environmental stress. Additionally, microarray data can be used to distinguish between
neutral and adaptive evolutionary processes affecting gene expression (e.g. Gibson, 2002;
Feder & Walser, 2005; Whitehead & Crawford, 2006). Nevertheless, a sequencing revolution
is currently driven by new technologies, collectively referred to as either ‘next-generation’
sequencing, ‘highthroughput’ sequencing, ‘ultra-deep’ sequencing or ‘massively parallel’
sequencing. These technologies allow us the large scale generation of ESTs efficiently and
cost-effectively available at the National Centre Biotechnology Information database [NCBI-
dbEST] (http://www.ncbi.nlm.nih.gov/dbEST); Shendure et al., 2005). There are increasing
studies in which 454 technologies, combined or not with Solexa/Illumina, are used to
characterize transcriptomes in several plant and animal species (Emrich et al., 2007;
Metzker, 2010; Eveland et al., 2008; Bellin et al., 2009). To give an idea of the potential
implications of these sequencing technologies it is enough to know that the pyrosequencing
delivers the microbial genome sequence in 1 hour, thus upsetting perspectives in basic
research, phylogenetic analysis, diagnostics as in industrial applications (Clarke, 2005;
Hamady et al., 2010; Yang et al., 2010; Claesson et al., 2009). Even in full sequenced
genomes, such as in Arabidopsis or humans, this deep sequencing is allowing to identify new
transcripts not present in previous ESTs collections (Weber et al., 2007; Sultan et al., 2010).
Also specific transcriptomes are being generated in species for which previous genomic
resources lacked because of the large size of their genomes (Alagna et al., 2009; Wang et al.,
2009; Craft et al., 2010) The new transcripts are also being used for microarrays design
(Bellin et al.,2009), and also for high throughput SSRs or SNPs identification. SNP detection
is performed by aligning raw reads from different genotypes to a reference genome or
transcriptome previously available in plants (Barbazuk et al., 2006), as in plants, (Trick et al.,
2009; Guo et al., 2010), animals (Satkoski et al., 2008) and humans (Nilsson et al., 2004).
De novo assembly of raw sequences coming from a set of genotypes, followed by pairwise
comparison of the overlapping assembled reads has also successfully used in species lacking
any significant genomic or transcriptomic resources (Novaes et al., 2008). The principle
behind these applications (as termed sequence census methods) is simple: complex DNA or
RNA samples are directly sequenced to determine their content, without the requirement
30 Systems and Computational Biology – Bioinformatics and Computational Modeling

for DNA cloning. Thus, these novel technologies allow the direct and cost-effective
sequencing of complex samples at unprecedented scale and speed, making feasible to
sequence not only genomes, but also entire transcriptomes expressed under different
conditions. Moreover, a unique feature of sequence census technologies is their ability to
identify, without prior knowledge, spliced transcripts by detecting the presence of sequence
reads spanning exon-exon junctions. Hence, next-generation sequencing delivers much
more information at affordable costs, which will increasingly supersede microarray based
approaches (Marguerat et al., 2008).
It is noteworthy, however, that transcription profiling has been questioned as an effective
tool for the discovery of genes that are functionally important and display variable
expression (e.g. Feder & Walser, 2005). In fact, the vast majority of genes implicated by
transcriptomics can be expected to have no phenotype. Furthermore, even if the synthesis of
mature protein is closely linked to the abundance of its corresponding mRNA, the
concentration of mature protein is the net of its synthesis and degradation. Degradation
mechanisms and rates can vary substantially and lead to corresponding variation in protein
abundance (Feder & Walser, 2005). The physiological measurements of protein abundance
for selected gene candidate could be a valuable addition to pure transcriptomic studies
(Jovanovic et al., 2010).
It is reasonable that a method should measure the most relevant output of gene expression,
namely dependent changes in protein amounts from potential target genes. Moreover, to be
worthwhile, the method should be easy to use, fast, sensitive, reproducible, quantitative and
scalable, as several hundred proteins have to be tested. A technique that promises to fulfill
most of those criteria is proteomics which is experiencing considerable progress after the
massive sequencing of many genomes from yeast to humans for both basic biology and
clinical research (Tyers & Mann, 2003). For identifying and understanding the proteins and
their functions from a cell to a whole organism, proteomics is a necessity in the assortment
of –omics technologies.
Historically, the term proteome was coined by Mark Wilkins first in 1994 as a blend of
proteins and genome and Wilkins used it to describe the entire complement of proteins
expressed by a genome, cell, tissue or organism. Subsequently this term has been specified
to contain all the expressed proteins at a given time point under defined conditions and it
has been applied to several different types of biological systems (Doyle, 2011; Ioannidis,
2010; Heazlewood, 2011; Prokopi & Mayr, 2011; Wienkoop et al, 2010).
In a basic view, a cellular proteome is the collection of proteins found in a particular cell
type under a particular set of conditions such as differentiation stage, exposure to hormone
stimulation inside tissues or changing of physical parameters in an environment. It can also
be useful to consider an organism's complete proteome, which can be conceptualized as the
complete set of proteins from all of the various cellular proteomes. This is very roughly the
protein equivalent of the genome. The term "proteome" has also been used to refer to the
collection of proteins in certain sub-cellular biological systems. For example, all of the
proteins in a virus can be called a viral proteome. The proteome is larger than the genome,
especially in eukaryotes, in the sense that there are more proteins than genes. This is due to
alternative splicing of genes and post-translational modifications like glycosylation or
phosphorylation. Moreover the proteome has at least two levels of complexity lacking in the
genome. When the genome is defined by the sequence of nucleotides, the proteome cannot
Bioinformatics Applied to Proteomics 31

be limited to the sum of the sequences of the proteins present. Knowledge of the proteome
requires knowledge of the structure of the proteins in the proteome and the functional
interaction between the proteins.
The escalating sequencing of genomes and the development of large EST databases have
provided genomic bases to explore the diversity, cellular evolution and adaption ability of
organisms. However, by themselves, these data are of limited use when they try to fully
understand processes such as development, physiology and environmental adaptation.
Taking advances from genomic information the proteomics can assign function to proteins
and elucidate the related metabolism in which the proteins act (Costenoble et al., 2011; Chen
et al., 2010; Joyard et al., 2010, Tweedie-Cullen & Mansuy, 2010).
In a wide-ranging functional view, proteomics is matching to genomics: through the use of
pure genome sequences, open reading frames (ORFs) can be predicted, but they cannot be
used to determine if or when transcription takes place. Proteomics, indicating at what level
a protein is expressed, can also provide information about the conditions under which a
protein might be expressed, its cellular location (Agrawal et al., 2010; Jamet et al., 2006;
Rossignol et al., 2006; Tyers & Mann, 2003), the relative quantities (Yao et al., 2001; Molloy et
al., 2005), and what protein–protein interactions take place (Giot et al., 2003; Schweitzer et
al., 2003). Genomics, in essence, demonstrates which genes are involved, whereas
proteomics can show clearer relationships by illustrating functional similarities and
phenotypic variances.
Because the environments in which organisms live is dynamic, the success of a species
depends on its ability to rapidly adapt to varying limiting factors such as light (for plants
above all), temperature, diet or nutrient sources. Since the proteome of each living cell is
dynamic, proteomics allows investigators to clarify if and to what extent various pathways
are utilized under varying conditions, triggered by the action of the environment on the
system, and relative protein-level response times. In other words how organisms are able to
biochemically survive to conditions imposed by environment.
Huge amount of data have been accumulated and organized in world-wide web sites served
for proteomics as main proteomics-related web sites have been lunched (Tab 1).
For example The Human Protein Reference Database represents a centralized platform to
visually illustrate and integrate information pertaining to domain architecture, post-
translational modifications, interaction networks and disease association for each protein in
the human proteome; on the ExPASy Proteomics site, tools are available locally to the server
or are developed and hosted on other servers.
As concerning plant proteomics, the research community is well served by a number of
online proteomics resources that hold an abundance of functional information. Recently,
members of the Arabidopsis proteomics community involved in developing many of these
resources decided to develop a summary aggregation portal that is capable of retrieving
proteomics data from a series of online resources (Joshi et al., 2010, http://gator.masc-
proteomics.org/ ). This means that information is always up to date and displays the latest
datasets. The site also provides hyperlinks back to the source information hosted at each of
the curated databases to facilitate analysis of the primary data. Deep analyses have also
performed on organelle proteomics as in protists, animals and plants. A well-known
database, launched in 2004, is devoted to proteomics of mitochondria in yeast (Ohlmeier et
al., 2004; http://www.biochem.oulu.fi/proteomics/ymp.html), while the Nuclear Protein
Database [NPD] is a curated database that contains information on more than 1300
32 Systems and Computational Biology – Bioinformatics and Computational Modeling

Table 1. List of the main proteome databases.

Bioinformatics Applied to Proteomics 33

vertebrate proteins that are thought, or are known, to localize to the cell nucleus. The database
can be accessed at http://npd.hgu.mrc.ac.uk and is updated monthly. Very recently, plant
organelle proteomics has experienced a rapid growth in the field of functional proteomics (see
the review, Agrawal et al., 2010); from this efforts gave rise seven main websites of which two
are devoted to the plastid (Plant Proteomic DataBase [PPDB] http://ppdb.tc.cornell.edu/),
two are specific for mitochondria (Arabidopsis Mitochondrial Protein DataBase [AMPDB],
http://plantenergy.uwa.edu.au/application/ampdb/; Arabidopsis Mitochondrial Protein
Project [AMPP], http://gartenbau.unihannover.de/genetic/AMPP), one is
an accurate database of comprehensive chloroplast proteome (AT_Chloro,
http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/).
An area of study within proteomics is ‘expression proteomics’, which is defined as the use of
quantitative protein-level measurements of gene expression to characterize biological
processes and deduce the mechanisms of gene expression control. Expression proteomics
allows researchers to obtain a quantitative description of protein expression and its changes
under the influence of biological perturbations, the occurrence of post-translational
modifications and the distribution of specific proteins within cells (Baginsky et al., 2010;
Roth et al., 2010).
As an example of high technological potential of expression proteomics, in the last ten years
plant proteomics research has been conducted in several land species achieving a high
degree of knowledge of the dynamics of the proteome in many model plants (Agrawal &
Rakwal, 2005; Baerenfaller et al., 2008; Grimplet et al., 2009; Komatsu, 2008; Plomion et al.,
2006) and thereafter translating this knowledge in other species whose genome sequence is
still under construction. The most successful studies are those which use separation of
subcellular compartments (Haynes & Roberts, 2007; Dunkley et al., 2006; Agrawal et al.,
2010) such as mitochondria (Heazlewood et al., 2005), chloroplast (Ferro et al., 2010),
endoplasmic reticulum (Maltman et al., 2007), peroxisomes (Fukao et al., 2002),
plastoglobules (Grennan, 2008), vacuoles (Jaquinod et al., 2007), nucleus (Repetto et al.,
2008) since they contain a limited number of proteins thus helping the protein identification.
Since 30 years, the greater part of research into the plant proteome has utilized two-
dimensional sodium dodecyl sulphate–polyacrylamide gel electrophoresis (2D SDS–PAGE)
for the protein separation step, which is usually followed by protein identification by mass
spectrometry (MS). Proteomics, the study of the proteome, has largely been practiced
through the separation of proteins by two dimensional gel electrophoresis. In the first
dimension, the proteins are separated by isoelectric focusing, which resolves proteins on
the basis of charge. In the second dimension, proteins are separated by molecular weight
using SDS-PAGE. The gel is dyed to visualize the proteins and the spots on the gel are
proteins that have migrated to specific locations.
The number of spots resolved in plant proteomics 2D projects depends on the chosen tissue
and plant species as well as the protein nature (i.g. basic or acid, soluble or membrane-
associated; Tsugita & Kamo, 1994; Porubleva et al., 2001). The gel plugs, containing the
proteins of interest are collected to further analyses by mass MS approaches and database
searches (Chevalier, 2010; Yates et al., 2009; Zhao &d Lin, 2010). This method where proteins
are analyzed after enzymatic digestion is widely used for high complexity samples in large
scale analyses and it is known as “bottom up approach” that was discussed in detail in the
next paragraph. Attention must given to the importance of sound statistical treatment of the
resultant quantifications in the search for differential expression. Despite wide availability of
34 Systems and Computational Biology – Bioinformatics and Computational Modeling

proteomics software, a number of challenges have yet to be overcome regarding algorithm

accuracy, objectivity and automation, generally due to deterministic spot-centric approaches
that discard information early in the pipeline, propagating errors. We review recent advances
in signal and image analysis algorithms in 2-DE, MS, LC/MS and Imaging MS.

Fig. 1. Proteome workflow I: after sample preparation and protein extraction, proteins are
initially separated by isoelectric focusing (IEF) in which they migrate along an IEF strip
which has a pH gradient between a cathode and an anode; the migration of each protein
ends when it reaches its isoelectric point in the gradient. This strip is then applied to a SDS
polyacrylamide gel in which the second dimension of the separation occurs according to
molecular weights. After fixation, the gel is stained by different techniques and its digital
image is acquired to be further analyzed by specific softwares, in order to found the
significant differentially expressed proteins.
With permission of Nova Science Publishers, Inc.
Bioinformatics Applied to Proteomics 35

3. Bioinformatics in proteomics
Mass spectrometry became a very important tool in proteomics: it has made rapid
progresses as an analytical technique, particularly over the last decade, with many new
types of hardware being introduced (Molloy et al, 2005; Matthiesen and Jensen, 2008, Yates
et al, 2009). Moreover, constant improvements have increased the levels of MS sensitivity,
selectivity as well as mass measurement accuracy. The principles of mass spectrometry can
be envisaged by the following four functions of the mass spectrometer: i) peptide ionization;
ii) peptide ions analyses according to their mass/charge ratio (m/z) values ; iii) acquisition of
ion mass data ; iv) measurement of relative ion abundance. Ionization is fundamental as the
physics of MS relies upon the molecule of interest being charged, resulting in the formation
of positive ions, and, depending on the ionization method, fragment ions. These ion species
are visualized according to their corresponding m/z ratio(s), and their masses assigned.
Finally, the measurement of relative ion abundance, based on either peak height or peak
area of sample(s) and internal standard(s), leads to a semi-quantitative request.

3.1 Typical procedure for proteome analysis

Proteome data elaboration procedure is different depending of the study target. In general
the studies can be qualitative in order to characterize the organisms expressed proteome and
quantitative to detect potential biomarker related to disease or other organism proprieties.
The principal proteomics studies are:
i. Full proteomics (qualitative);
ii. Functional proteomics (relative quantitation studies);
iii. Post translational modification functional proteomics (qualitative and relative
quantitation studies)

3.2 Data elaboration for full proteome analysis

In full proteomics analysis (Armengaud et al. 2010) the proteins are usually extracted and
qualitatively identified. These studies are usually performed in order to understand what
proteins are expressed by the genome of the organism of interest. The general analytical
scheme is reported in Figure 2.
Basically, after protein separation, mainly through gel electrophoresis or other separation
approaches (liquid chromatography etc.), proteins are identified by means of mass
spectrometric technique. Two kind of data processing algorithms can be employed
depending by the analytical technology used to analyze the proteins. The two approaches
are:
i. Bottom up approach. It is used to identify the protein of interest after enzymatic or
chemical digestion;
ii. Top down approach. In this case proteins are not digested but directly analyzed by
mass spectrometric approaches;
In the former case (bottom up) the protein are digested by means of enzymatic or chemical
reaction and the specific peptides produced are then analyzed to identify the protein of
interest. This results can be obtained using mass spectrometric mass analyzer that can
operate in two conditions: a) full scan peptide mass fingerprint (MS) and b) tandem mass
spectrometry (MS/MS). In the case a) the mass/charge (m/z) ratio of the peptide is
obtained using high resolution and mass accurate analyzer (time of flight, FTICR; see
36 Systems and Computational Biology – Bioinformatics and Computational Modeling

Cristoni et al., 2004, 2003). The combination of the high accurate m/z ratio of the detected
peptides is checked against the theoretical one generated by virtual digestion of the proteins
present in the known database. A list of protein candidates is so obtained with relative
statistical identification score, correlated to the number of peptides detected, per proteins
and peptide mass accuracy. The principal software package used for this kind of data
elaboration are reported in table 2.

Fig. 2. General analytical scheme of Full proteomic analysis.

Bioinformatics Applied to Proteomics 37

Software name Availability Web Categor

Aldent
fre http://www.expasy.org/tools/aldente/ database searching and PMF
FindPep
Open source,free http://www.expasy.ch/tools/findpept.html database searching and PMF
FindMo
Open source,free http://www.expasy.ch/tools/findmod database searching , PM and
MASCO
Commercial http://www.matrixscience.com database searching and PMF
ProFoun Open source,free http://prowl.rockefeller.edu/prowl-cgi/profound.exe database searching and PMF
PepNovo
Ope source,free http://peptide.ucsd.edu/pepnovo.py DeNov
PEAK
Commercial http://www.bioinfor.com/peaksonline DeNov
Lutefis
Open source,free http://www.hairyfatguy.com/Lutefisk/ DeNov
SEQUES
Commercial http://fields.scripps.edu/sequest databas searchin
XTandem
Open source,free http://www.thegpm.org/tandem database searching
OMSS
Open source,free http://pubchem.ncbi.nlm.nih.gov/omssa database searching
PHENY
Commercial http://www.phenyx-ms.co database searching
ProbI
Open source,free http://www.systemsbiology.org/research/probid/ database searching
Popita
Open source,free http://www.expasy.org/people/pig/heuristic.html database searching
Interac
Open source,free http://www.proteomecenter.org/software.php database searching
DTAselec
Open source,free http://fields.scripps.edu/DTASelect/index.html database searching
Chompe
Open source,free http://www.ludwig.edu.au/jpsl/jpslhome.html database searching
ProteinProphet
Open source,free http://www.proteomecenter.org/software.php database searching
FindPep
Open source,free http://www.expasy.ch/tools/findpept.html database searching
GutenTa
Open source,free http://fields.scripps.edu/ Denovo and PTM
M -Convolution
fre DeNov and
http://prospector.ucsf.edu/prospector/mshome.htm
M -Alignment
fre DeNov and
http://prospector.ucsf.edu/prospector/mshome.htm

Table 2. Summary of the most recognized softwares employed for protein analysis.
For instance one of the most employed algorithm for PMF is Aldente (http://www.expasy.ch).
This software allows the protein identification in multi-step way. In the first step the most
statistically significant proteins are identified on the basis of accurate peptide m/z
combination. In the second one the peptide m/z ion leading to the first identification are not
considered and other spectra m/z signal combination are considered in order to identify other
proteins. The step is reaped since the identification statistic is good enough in order to identify
the protein candidates. In the case b) (MS/MS) the peptides are fragmented using different
kind of chemical physical reactions [collision induced dissociation (CID), electron transfer
dissociation (ETD), ecc]. The m/z ratio of the peptide fragments is then analyzed in order to
obtain peptide structural information. Two approaches are usually employed in order to
elaborate the fragmentation spectra: database search and de novo sequence. In the case of
database search, the peptide MS/MS fragmentation spectra are matched against the theoretical
one extracted from public or private repositories. The peptide sequence identification is
obtained on the basis of a similarity score among the experimental MS/MS and the theoretical
MS/MS spectra. The main limitation of this approach is that only known proteins, reported in
the database can be identified. For instance, Thegpm (The Global Proteome Machine;
http://www.thegpm.org) is an open source project aims to provide a wide range of tools for
proteome identification. In particular X!Tandem software (Muth et al., 2010) is widely
employed for database search protein identification. When the protein sequence is not
perfectly known, denovo sequence method can be used. In this case, the sequence is obtained
directly from the MS/MS spectra avoiding the step of database spectrum search. The obtained
38 Systems and Computational Biology – Bioinformatics and Computational Modeling

sequences are then compared with those contained in the database so to detect homologies.
Even in this case a statistical protein identification score is calculated on the basis of the
number of homologues fragments obtained for each protein candidate. The software usually
employed in the case of database search approach are classified in table 2 together with those
employed for de novo. An example of software used for de novo porpoises is PepNovo (Frank
et al., 2005). It has been presented a novel scoring method for de novo interpretation of
peptides from tandem mass spectrometry data. Our scoring method uses a probabilistic
network whose structure reflects the chemical and physical rules that govern the peptide
fragmentation. The algorithm was tested on ion trap data and achieved results were
comparable and in some cases superior to classical database search algorithms. Moreover,
different elaborative approaches have been developed in order to increase the sample
throughput and statistical accuracy of the identification process (Jacob et al., 2010). Various
statistical validation algorithms have been translated into binary programs and are freely
distributed on the internet (table 1). Others are not freely available while some have been
theoretically described but have not been translated into a freely available or commercial
binary program (table 1). It must be stressed that open-source and freely available programs
are capable of highly accurate statistical analysis. For example, an interesting free program
referred to as ProbID (Zhang et al., 2002) is freely available for evaluation. This program is
based on a new probabilistic model and score function that ranks the quality of the match
between the peptide. ProbID software has been shown to reach performance levels
comparable with industry standard software. A variety of other software, based on heuristic or
similar to ProbID Bayesian approach have been developed (Jacob et al., 2010). Some of these
software are reported in table 2. It must be stressed that many of these software packages
require a web server to operate (e.g., ProbID). This fact introduces some problems related to
the difficulty to administrate a server, especially from a security point of view in the case of
cracker informatic attacks to a chemstation connected to the internet.
The analysis of intact proteins (top down approach) can be an alternative to bottom up one
(Cristoni et. al., 2004). In the first step of data elaboration, the molecular weight of the
protein is obtained using dedicated deconvolution algorithm. For instance, Zheng and
coworkers have proposed a new algorithm for the deconvolution of ESI mass spectra based
on direct assignment of charge to the measured signal at each m/z value in order
consequently indirectly to obtain the protein molecular weight (Zheng H, et al. 2003).
Another interesting deconvolution approaches is based on the free software named MoWeD
(Lu et al., 2011). It can be used to rapidly process LC/ESI/MS data to assign a molecular
weight to peptides and proteins. It must be stressed that, the list of found components can
also be compared with a user defined list of target molecular weight values making it easy
to identify the different proteins present in the analyzed samples. However, when the
protein sample mixture is highly complicated, these software tools could fail. This occurs
especially if the analysis is performed using low mass accuracy instruments (e.g., IT) and if
the chromatographic separation performed before MS analysis is not optimal. Thus, the
molecular weight data must be integrated with protein sequence information. In this case,
intact proteins ions are analyzed and fragmented by means of high resolution and mass
accuracy mass analyzer (e.g.: FTICR, orbitrap, QTOF etc;). The mass spectra obtained are
matched directly with the theoretical one present in the database and a statistical score,
based on the spectra similarity, is associated with the protein identification. The main
advantage of this technology is the ability to investigate intact proteins sequence directly
avoiding time consuming digestion steps. On the other hand the majority of algorithm are
Bioinformatics Applied to Proteomics 39

usually developed for bottom up approach. In fact for different chemical physical reasons,
that are not related to this chapter theme, the sensitivity in detecting high molecular weight
proteins is definitely lower with respect to that obtained by detecting low molecular weight
peptide after protein digestion. An example of algorithm for protein identification, by intact
protein ion fragmentation, has been proposed by McLafferty and co-workers (Sze et al.,
2002). A free web interface to be used to analyze proteins MS data using the top-down
algorithm is available free of charge for academic use. In the top-down MS approach, the
multicharged ions of proteins are dissociated and the obtained fragment ions are matched
against those predicted from the database protein sequence. This is also a very powerful tool
to characterize proteins when complex mixtures are available.

3.3 Data elaboration for functional proteome

Functional proteome (May et al., 2011) is related to both identify differentially expressed
proteins among different sample lines and obtain their relative quantitation. For instance, it
is possible to compare differentially expressed proteins among control and unhealthy
subjects affected by different diseases (Nair et al., 2004). The classical approach (Figure 3) is
based on the protein separation by means of 2D-GEL electrophoresis.
The protein are then colored by using specific reagent (e.g. blue coumassie, silver stain etc)
and the gel images are obtained by means of a normal or laser fluorescence scanner. Specific
software are then employed in order to overlap the images and detect the differentially
expressed proteins on the basis of the color intensities. This approach, has strong limitations
mainly in terms of elaboration time needed to obtain the match. Nowadays some apparatus
have been developed in order to mark, with different label fluorescence reagents, the
proteins extracted from different spots. Thus it is possible to run more samples at the same
time and detect the proteins of more spots, separately, by means of different fluorescence
laser. Distinctive images relative to different gradient of fluorescence are so simultaneously
obtained, this results in differentially expressed proteins.
High innovative shut-gun technology based on liquid chromatography coupled to high
resolution mass spectrometry, have been recently developed and employed for functional
proteomics purposes. In particular, to compare a complex protein mixture of different
experimental lines, the obtained peptides after digestion have been analyzed by means of
Surface Activated Chemical Ionization (SACI; Cristoni et al. 2007) technologies coupled to
high relation and mass accuracy mass analyzer (e.g. Orbitrap, QTOF etc). Very recently
SACI technology has been applied in seagrass proteomics (Finiguerra et al., 2010). In fact,
the increasing sensitivity of this ionization device improves peptides detection thus
recovering the limited sea grass genome resources. SACI leads to benefits, in complex plant
protein mixture analysis, in terms of quantitative accuracy, precision, and matrix effect
reduction, that have been widely demonstrated (Cristoni et al., 2009). As regard peptide
analysis, it was observed that, by changing in-source ionization conditions, one could
selectively produce both in-source singly and doubly charged species (Cristoni et al., 2007),
which are both of interest. This technologic approach yields maximum benefits when data
are acquired using a high mass-accuracy and high-resolution mass analyzer that can operate
in both full-scan and tandem mass spectrometry (MS/MS) acquisition conditions. The SACI
technique strongly increased the number of detectable proteins and of assigned peptides for
each protein. For example, with SACI technology application, it was possible to identify a
previously identified protein (a heat shock cognate protein), 1000 fold over expressed in
40 Systems and Computational Biology – Bioinformatics and Computational Modeling

deeper plants (-27 m) in comparison with the more shallow plants (-5m), detecting four
peptides respect to only two detected by micro-ESI (Finiguerra et al., 2010).

Fig. 3. Classical approach for functional proteome analysis.

Bioinformatics Applied to Proteomics 41

The differentially expressed peptides are compared using specific chromatographic

alignment software (Sandin et al., 2011). One of the most effective software is named XCMS
(Margaria et al., 2008; Kind et al., 2007). The peptide mass fingerprint of the differentially
expressed peptides followed by database search and de novo sequencing approach lead to
the rapid identifications of differentially expressed proteins.

3.4 Data elaboration for the study of post translational modification

Post translational modification (PTM) detection and quantization is one of the most difficult
task in proteomics research. Usually, they are detected through bottom up approaches. For
example, considering that phosphorylated peptides do not show a high quality signal
intensity, consequently leading to lower sensitivity, dedicated informatics tools have been
developed in order to characterize the phosforilation sites on the basis of peptides
fragmentation spectra. Different tools developed for this purpose are reported in table 1. All
tools detect the modified sites on the basis of fragmentation spectra. Basically, the MS/MS
spectra of the theoretical modified peptides are calculated and matched with the
experimental one. Even in this case the similarity score is used in order to identified the
peptides and the corresponding phosphorylation site.
In the case of PTM the classical PMF and database search approaches cannot be used due to
the fact that the modifications and the mutations cause shifts in the MS/MS peaks. Several
approaches use an exhaustive search method to identify and characterize the mutated
peptides. A virtual database of all modified peptides for a small set of modifications is
generated and the peptide MS/MS spectrum is matched against the theoretical spectra of
the virtually modified peptides. However, the MS/MS peak shifts result in an increase in
the search space and a long elaboration time could be required. To solve this problem some
algorithms have been developed (Cristoni S, Bernardi LR. et al. 2004). A good method to
detect the mutated peptides is based on the tags approach. For example, the GutenTag
software developed by Yates and coworkers use this strategy to identify and characterize
the mutated peptides (Tabb DL, et al. 2003). This software infers partial sequences (‘tags’)
directly from the fragment ions present in each spectrum, examines a sequence database to
assemble a list of candidate peptides and evaluates the returned peptide sequences against
the spectrum to determine which is the best match. The software, written in the Java
programming language, runs equally well under Microsoft Windows, Linux and other
operating systems. GutenTag is specific to doubly charged peptides. Pevzner and coworkers
have also developed some interesting algorithms for the analysis of peptide mutations
(Pevzner PA, et al. 2001). In this case, two software packages (MS-CONVOLUTION and MS-
ALIGNMENT) that implement the spectra (table 2) convolution and spectral alignment
approaches, to identify peptides obtained through enzymatic digestion, have been used to
identify and characterize peptides differing by up to two mutations/modifications from the
related peptide in the database. This is a two-stage approach to MS/MS database searching.
At the first stage, the spectral alignment is used as a filter to identify t, top-scoring peptides
in the database, where t is chosen in such a way that it is almost guaranteed that a correct hit
is present among the top t list. These top t hits form a small database of candidate peptides
subject to further analysis at the second stage. At the verification stage, each of these t
peptides can be mutated (as suggested by spectral alignment) and compared against the
experimental spectrum. However, the peptide mutation or modification can produce low
informative fragmentation behavior (Cristoni et al., 2004), in which case the protein
modification identification may fail. It is also possible to use the PMF approach to
42 Systems and Computational Biology – Bioinformatics and Computational Modeling

characterize mutations and modifications (Cristoni et al., 2004). In this case, it is necessary to
use a high mass accuracy mass spectrometer since the characterization of a mutation or
modification is based on the identification of the accurate m/z ratios of digested peptide.
Freeware software to identify protein modifications and mutations using database search
and PMF are reported in the Information Resources section. For example, the software
FindPept is capable of identifying peptides that result from nonspecific cleavage of proteins
from their experimental masses, taking into account artifactual chemical modifications, PTM
and protease autolytic cleavage. If autolysis is to be taken into account, an enzyme entry
must be specified from the drop-down list of enzymes for which the sequence is known.
Furthermore, this is a web application installed on the expasy website and therefore it is not
necessary to install and administrate it on a local server. Another field in which different
algorithms have been employed is the characterization of disulfide cross-link locations
(Cristoni et al., 2004). For instance, some tools available on a public website
http://www.expasy.ch were recently developed for this purpose. This software is referred
to as Protein Disulfide Linkage Modeler and it permits the rapid analysis of mass spectromic
disulfide cross-link mapping experiments. The tool can be used to determine disulfide
linkages in proteins that have either been completely or partially digested with enzymes.
The masses of all possible disulfide-linked multichain peptide combinations are calculated
from the known protein sequence and compared with the experimentally determined
masses of disulfide-linked multichain peptides. Thus, this software is based on the
fragmentation behavior of the cross-linked peptides obtained by enzymatic digestion.
However, several issues may occur despite the fact that this algorithm executes its work
very well. The major issue is that proteins containing linker cysteines have domains that are
very resistant to proteolysis. Furthermore, the fragmentation of the cross-linked peptide ions
may lead to a spectra that is difficult to elaborate even if specific algorithms are used. This is
due to the high chemical noise level that is present in the fragmentation spectra of their
multicharged ions (Craig et al., 2003).

4. Data management - The advent of semantic technologies and machine

learning methods for proteomics
For Systems Biology the integration of multi-level Omics profiles (also across species) is
considered as central element. Due to the complexity of each specific Omics technique,
specialization of experimental and bioinformatics research groups have become necessary,
in turn demanding collaborative efforts for effectively implementing cross-Omics
(Wiesinger M, et al. 2011).
In recent years large amounts of information have been accumulated in proteomic, genetic
and metabolic databases. Much effort has been dedicated to developing methods that
successfully exploit, organize and structure this information. In fact semantic is the study of
meaning. In the case of proteomics it can be used in order to find specific relations among
proteins and metabolomics, genomics and ionomics networks. For instance the group of
Masaneta-Villa and co-workers (Massanet-Vila et al., 2010) has developed a high-
throughput software package to retrieve information from publicly available databases,
such as the Gene Ontology Annotation (GOA) database and the Human Proteome Resource
Database (HPRD) and structure their information. This information is presented to the user
as groups of semantically described dense interaction subnetworks that interact with a
target protein. Another interesting technology in the semantic field has been proposed by
Bioinformatics Applied to Proteomics 43

the group of Mewes HW. and co-workers (Mewes et al., 2011). This group has many years
of experience in providing annotated collections of biological data. Selected data sets of high
relevance, such as model genomes, are subjected to careful manual curation, while the bulk
of high-throughput data is annotated by automatic means. This is, in fact an important
point, manual curation is essential for semantic technology purposes. The data mean must
be carefully checked before of the insertion in the semantic database otherwise serious
meaning error can occurs during the research phase. High-quality reference resources
developed in the past and still actively maintained include Saccharomyces cerevisiae,
Neurospora crassa and Arabidopsis thaliana genome databases as well as several protein
interaction data sets (MPACT, MPPI and CORUM). More recent projects are PhenomiR, the
database on microRNA-related phenotypes, and MIPS PlantsDB for integrative and
comparative plant genome research. The interlinked resources SIMAP and PEDANT
provide homology relationships as well as up-to-date and consistent annotation for
38,000,000 protein sequences. PPLIPS and CCancer are versatile tools for proteomics and
functional genomics interfacing to a database of compilations from gene lists extracted from
literature. A novel literature-mining tool, EXCERBT, gives access to structured information
on classified relations between genes, proteins, phenotypes and diseases extracted from
Medline abstracts by semantic analysis.
Another interesting semantic application has been shown by Handcock J. and co-workers
(Handcock, et al., 2010). This group has semantically correlate proteomics information to
specific clinical diseases. They have produced a database mspecLINE. Given a disease, the
tool will display proteins and peptides that may be associated with the disease. It will also
display relevant literature from MEDLINE. Furthermore, mspecLINE allows researchers to
select proteotypic peptides for specific protein targets in a mass spectrometry assay.
Another interesting semantic technology is based on machine learning and is employed for
biomarker discovery purposes (Barla et al., 2008).The search for predictive biomarkers of
disease from high-throughput mass spectrometry (MS) data requires a complex analysis
path. Preprocessing and machine-learning modules are pipelined, starting from raw spectra,
to set up a predictive classifier based on a shortlist of candidate features. As a machine-
learning problem, proteomic profiling on MS data needs caution like the microarray case.
The risk of over fitting and of selection bias effects is in fact, pervasive.
Summarizing semantic technologies can be useful both to correlate the different omics
sciences information and to correlate the single omics (e.g. proteomics) to specific
information like clinical disease correlated to differentially expressed proteins between
control and unhealthy groups (biomarker discovery).

5. Conclusions
Bioinformatics for proteomics has grown significantly in the recent years. The ability of
process an high amount of data together with the high specificity and precision of the new
algorithm in the protein identification, characterization and quantization make now possible
to obtain an high amount of elaborated data.
The main problem remain the data management of a so high amount of data. Find the
correlation among different proteomic data and the other omics sciences (metabolomics,
genomics, ionomics) still remain a difficult task. However, database technology together
with new semantic statistical algorithm are in evolution powerful tools useful to overcome
this problem.
44 Systems and Computational Biology – Bioinformatics and Computational Modeling

6. Acknowledgment
This is in memory of Anna Malomo-Mazzuca and Remo Cristoni, mother and father of the
authors.

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