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Proteogenomic Mapping for Structural Annotation of Prokaryote Genomes

Nan Wang Shane Burgess, Mark Lawrence

School of Computing Department of Basic Sciences
University of Southern Mississippi Mississippi State University
Hattiesburg, MS 39406, USA Mississippi State, MS 39762, USA
Nan.Wang@usm.edu burgess@cvm.msstate.edu

Susan Bridges
Department of Computer Science and Engineering
Mississippi State University
Mississippi State, MS 39762, USA
bridges@cse.msstate.edu

Abstract——Structural annotation of genomes is one of I. INTRODUCTION

major goals of genomics research. Most popular tools The explosion in genomic sequencing has
for structural annotation of genomes are determined by
led to the availability of a large number of genomes
computational pipelines. It is well-known that these
computational methods have a number of shortcomings over the last decades and these genome sequences are
including false identifications and incorrect now publicly available [1]. However, the genome
identification of gene boundaries. Proteomic data can sequences by themselves are of little use. True value
used to confirm the identification of genes identified by is derived from the genome sequence only after the
computational methods and correct mistakes. A genes have been identified (structural annotation) and
Proteogenomic mapping method has been developed, the function of the protein product has been
which uses peptides identified from mass spectrometry determined (functional annotation). After a genome
for structural annotation of genomes. Spectra are has been sequenced, gene prediction programs are
matched against both a protein database and the
used to predict genes. Most computational programs
genome database translated in all six reading frames.
Those peptides that match the genome but not the for structural annotation in bacteria are based on
protein database potentially represent novel protein features in the nucleic acid sequence. GeneMark [2]
coding genes, annotation errors. These short and Glimmer [3] are popular gene finding tools, and
experimentally derived peptides are used to discover both are based on Hidden Markov models (HMMs) at
potential novel protein coding genes called expressed the nucleic acid sequence level. Although these tools
Protein Sequence Tags (ePSTs) by aligning the peptides are widely used, they are known to have a number of
to the genomic DNA and extending the translation in shortcomings including failing to identify genes that
the 3' and 5' direction. In the paper, an enhanced exist, false identifications, and incorrect identification
pipeline, has been designed and developed for
of gene boundaries [4]. Because of the high false
discovering and evaluating of potential novel protein
coding genes: 1) a distance-based outlier detection identification rate obtained for prediction of short
method for validating peptides identified from MS/MS, genes, these algorithms usually use a somewhat
2) a proteogenomic mapping for discovery of potential arbitrary length cutoff and are therefore particularly
novel protein coding genes, 3) collection of evidence ineffective at identifying novel short genes.
from a number of sources and automatically evaluate Mass spectrometry [5] is a popular
potential novel protein coding genes by using machine technique for detection of proteins in biological
learning techniques, such as Neural Network, Support samples. It provides direct molecular evidence of the
Vector Machine, Naïve Bayes etc. existence of the protein in the living cell. Proteins are
Keywords-peptide validation; outlier detection; ePST;
typically identified using mass spectrometry by
expressed Protein Sequence Tags; proteogenomic computationally matching experimental mass spectra
mapping; neural network; support vector machine; naïve against theoretical spectra derived from a protein
Bayes; Bayesian network; potential genes; target decoy database. However, several groups have recently
strategy reported the use of mass spectral data to identify

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genes on the genome [4]. This process was named
proteogenomic mapping by Jaffe et al. [1]. We have
designed algorithms that use experimental protein
data from mass spectrometry to find novel protein
coding genes on bacterial genomes. Mass
spectrometry cannot be used to identify proteins
directly. Instead, the proteins are cleaved into
peptides by enzymes such as trypsin that cut the
protein in specific places. The peptides are assigned
to mass spectrometry and identified by matching the
resulting spectra against a database of theoretical
spectra. Once the peptides have been identified, they
are mapped to the parent protein. Peptide
identifications based on mass spectrometry are
extremely noisy and represent a mixture of true and
false identifications. PepOut [6], a distance-based
outlier detection method is used for peptide
validation. In the paper, we describe a proteomics
based method for identifying open reading frames
that are missed by computational algorithms. Mass
spectrometry based identification of peptides and
proteins from biological samples provide evidence
for the expression of the genome sequence at the
protein level. This proteogenomic mapping method
uses proteomics to both confirm computationally
predicted genes and to identify novel gene models.
II. METHOD
In the paper, we describe our proteogenomic
mapping pipelinea set of computational tools that
automates the structural annotation of bacterial
genomes using proteogenomic mapping pipeline. The
work flow is shown in Figure 1. There are four major
steps in the pipeline including peptide validation by
PepOut [6], Proteogenomic mapping for ePST
generation, feature collection from variety resources
and ePST evaluation by machine learning algorithms.
A. Proteogenomic mapping pipeline
To discovery potential novel protein coding genes,
a proteogenomic mapping pipeline was developed
shown in Figure 1. Biological samples are trypsin
digested to peptides. These samples are run in an LC
ESI-MS/MS mass spectrometer which generates mass
spectra for the samples. Mass spectra then are
searched against the protein database and the genome
database translated in six reading frames. Those
peptides that match the genome but not the protein
database potentially represent novel protein coding
genes or annotation errors, and are called as unique
peptides. The peptide identifications generated from
protein and genome database search results are
validated by PepOut. The unique peptides are used for
discovering the potential protein coding genes by
proteogenomic mapping, and these potential protein
coding genes are called as ePSTs (expressed Protein
Sequence Tags). These ePSTs are used further for
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correcting the annotation error/ sequencing error,
some might be novel protein coding genes. To
discover these unique peptides, the proteogenomic
mapping workflow requires a sequenced genome, the
existing protein models for the genome, and a
proteomics dataset which is specific to the prokaryotic
genome under study.
Figure 1. Flowchart of proteogenomic mapping pipeline.
B. Discovery of potential novel protenting
coding genes
Unique peptides are segments of expressed
protein sequences, and can be used to discover
potential protein coding genes in the genome. Figure
2 illustrates the process used to identify potential
novel genes or to correct predicted genes. Peptides
with high confidence scores from PepOut [6] are
used to discover expressed protein sequence tags
(ePSTs). The genome sequence is translated in all six
reading frames and the validated unique peptides are
mapped to the translated sequence and are assumed
to represent a segment of an expressed gene. The
next task is to find the beginning and end of the gene.
The peptide match is extended downsteam to find an
inframe stop codon. To find the start codon, we first
locate an inframe upstream stop codon. The start
position of a potential protein coding gene should be
located between the inframe stop codon of an
upstream gene and the beginning of the peptide. In
our method, we use the first start codon between the
upstream inframe stop codon and the beginning of
the peptide as the start codon of the potential protein
coding gene. If there is no start codon between the
inframe upstream stop codon and the peptide, the
beginning of the peptide is used as the start position
of the potential protein coding gene. The ePSTs
generated using the process described above are
considered potential novel protein coding genes or
extensions to predicted genes and are evaluated
further as described in the next sections.
 Column Name Description
ePST_length Number of amino acids in the ePST
sequence
ePST_probability Probability that the ePST is a true
identification based on the probability of the
peptides used to generate the ePST.
Number_peptides Number of peptides matched to the ePST
Coverage Percentage of amino acids in the ePST
covered by peptides. Multiple peptides
matching a single ePST may overlap.
Amino acids matching several peptides are
counted only one time.
StartCodon The start codon for the ePST. A value of ““-
““ means no start codon
NewStartCodon The start codon suggested by RBSFinder
RBS Pattern of the ribosomal binding site of the
ePST generated by RBSFinder
CDD Yes if the ePST has a conserved domain
identified by CDD
Homology protein Yes if the ePST has a homologous match in
match the protein database for a a related organism,
no otherwise. Additional information: hit
length, percent identity.
Homology DNA Yes if the ePST has a homologous match in
Figure 2. The process used to generate ePSTs from peptide
match the genome database for a related organism,
sequences generated from trandem mass spectra.
no otherwise. Additional information: hit
length, percent identity.
C. Potential gene feature collection

Due to noise from a variety of sources D. Evalutaion of potentail new genes

including the collection of biological samples, errors
associated with mass spectrometry, sequencing errors
etc, many of these potential new genes discovered by
proteogenomic mapping may be false positive
identifications. The most common method used for
validating potential novel protein-coding genes
predicted from expressed protein evidence is real-
time PCR. However, real-time PCR is an expensive
labor-intensive process and cannot be used for a large
number of potential candidate genes. Therefore, there
is a need to collect evidence of the coding potential
of ePSTs and to develop machine learning methods
for automatic evaluation. Biologists provided us with
a list of potentially useful features for evaluating
ePSTs. In this section, we will describe feature
information recommended by biologists for ePST
evaluation and describe how we derive this
information. Table 1 lists the features we extract for
each ePST that we will use to train our machine
learning models. Note that some of these entries
correspond to more than one ““feature.”” For example,
for each homology search, when there is a match we
also find the length of the match and the percent
identity. Values of 0 were used for both the length of
the match and the percent identity if there is no match.
The ePST with the features are ready to be validated.
Supervised machine learning algorithms
such as decision tree, support vector machine and
nerural network are used for training a model to
classifying the ePST as true or false novel genes. To
train a model, a subset of ePSTs with all appeared
feature combination is collected and the dataset is
evaluated by human expetises as true or false novel
genes.
III. RESULTS AND DISCUSSION
A. Experimenal datasets
Our experiments were done on Mannheimia
haemolytica. M. haemolytica has been the most
commonly isolated species. Dr. Sarah Highlander at
the Baylor College of Medicine directed genome
sequencing of M. haemolytica. A list of 2434
predicted genes are annotated names and unique
COG (clusters of orthologous group) numbers are
available at the Baylor College of Medicine M.
haemolytica genome web page. Currently, Dr.
Highlander has organized a multi-institutional effort
to annotate the M. haemolytica genome to
standardize its gene ontology and make the data more
useful to the BRD.
B. Results and discussions

TABLE I. FEATURES USED FOR EVALUATING THE SEQUEST [42] search algorithm is used for
IDENTIFICATION OF POTENTIAL NOVEL GENES identifying peptide-spectra matches. M.
haemolyticamass spectra are search against a protein
database provided by Dr. Highlander and a genome

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database translated in six reading frames. highest accuracy of the classifiers tested, the resulting
Preprocessing of the evaluation dataset was required model is difficult to understand. The support vector
because our features include a combination of real machine resulted in lower accuracy than other
numbers and categorical data. Numerical features classifiers. Rule based classifiers such as NNge
were discretized based on the experts’’ suggestions. (Non-Nested Generalized Exemplars) find a set of
The feature ““ePST length”” was discretized into three rules that can be used for classification. However, the
bins: 0 –– 50 aa, 51 - 100 aa, and > 100 aa, where aa is resulting rules can be quite complex and difficult to
the number of amino acids. The feature understand. Instance based learning algorithms such
ePST_probability was discretized into two bins: 0- as some nearest neighbor find the training instance
70% and 71%-100%. The feature peptide_coverage closest to the given test instance, and predicts the
is discretized into two bins: < 30% or • 30%. The same class as this training instance. These algorithms
feature ePST matches is divided into the categories: S may result in high accuracy but do not provide any
single match or M multiple matches. In the results, information about the knowledge domain.
3812 peptides were identified by PepOut with p > 0.5
and 3496 ePSTs were generated from 3812 peptides. IV. CONCLUSIONS
Software was developed to select a training subset
with all appeared possible combinations of feature Mass spectrometry provides an independent and
values that occur in the dataset where 10 examples complementary method for protein detection and
are selected for each possible combination. This allows one to confirm the computational annotated
resulted in a training set consisting of 117 of the 3496 proteins. Here we developed the proteogenomic
ePSTs generated. These 117 ePSTs in training data mapping pipeline to improve the structural annotation
samples were labeled as T (true gene) or F (false gene) of genomes, correct and confirm genome annotations,
by two experts in bacterial genomics (Dr. Bindu discover novel protein coding genes. Machine
nanduri and Dr. Mark Lawrence of the college of learning algorithms can be used to predeuct the
Veterinary Medicine). In the 117 training data confidence in identification of novel genes.
samples, there are 47 of 117 positives and 70 of 117 ACKNOWLEDGMENT
negatives. Weka [7] which is a datamining toolkit is
used for the experiments. Table II shows the accuracy The project was supported by the National
Research Initiative of the USDA Cooperative State
comparison of machine learning algorithms such as
Research, Education and Extension Service, grant
tree ––based alrogithms, support vector machine, rule-
number #2006-35600-17688. It was partially funded
based algorithms, and neural network. by a grant from the National Science Foundation
(EPS-0556308-06040293).
TABLE II. COMPARISON OF MACHINE LEARNING
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