Nucleic Acids Research Advance Access originally published online on November 4, 2008
Nucleic Acids Research 2009 37(Database issue):D181-D184; doi:10.1093/nar/gkn804
Nucleic Acids Research, 2009, Vol. 37, Database issue D181-D184
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
NOPdb: Nucleolar Proteome Database—2008 update
Yasmeen Ahmad1,
François-Michel Boisvert1,
Peter Gregor2,
Andy Cobley2 and
Angus I. Lamond1,*
1Wellcome Trust Centre for Gene Regulation & Expression and 2School of Computing, University of Dundee, Dundee DD1 5EH, UK
*To whom correspondence should be addressed. Tel: +44 1382 385473; Fax: +44 1382 345695; Email: angus{at}lifesci.dundee.ac.uk
Received September 12, 2008. Revised October 10, 2008. Accepted October 10, 2008.
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ABSTRACT
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An experimental data handling system has been created as an
update to the previous Nucleolar Proteome Database (NOPdb3.0:
http://www.lamondlab.com/NOPdb3.0/). This updated system is
able to manage large data sets identified by multiple mass spectrometry
and has been used to analyse highly purified preparations of
human nucleoli from different cell lines. The newly created
application includes a dynamic relational database, which is
kept up to date by laboratory staff. The data are further annotated
with information from specific external sources on the web,
including the IPI and Gene Ontology databases. In addition,
an Application Programming Interface provides external users
with a portal to link into the nucleolar proteome database and
hence, gain access to continually updated results. From the
initial
700 human proteins identified in the previous iteration
of the NOPdb, we have now identified over 50 000 peptides contained
in over 4500 human proteins from purified nucleoli, providing
enhanced coverage of the nucleolar proteome.
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INTRODUCTION
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The nucleolus is a highly conserved nuclear organelle whose
main function is to coordinate the synthesis and assembly of
ribosome subunits (
1). Previously we described a Nucleolar Proteome
Database (NOPdb2.0:
http://www.lamondlab.com/NOPdb) that archived
data on >700 proteins that were identified by multiple mass
spectrometry (MS) analyses from highly purified preparations
of human nucleoli (
2). Each protein entry was annotated with
information about its corresponding gene, its domain structures
and relevant protein homologues across species, as well as documenting
its MS identification history, including all the peptides sequenced
by tandem MS/MS. Moreover, data showing the quantitative changes
in the relative levels of approximately 500 nucleolar proteins
were compared at different time points upon transcriptional
inhibition (
3).
The data presented by the previous NOPdb, version 2.0, was held in a flat file database. Due to the aggregated nature of the data, results from individual experiments could not be extracted. The peptide data for a single protein were merged within this database rather than stored separately. The client interface to this database consisted of Perl CGI scripts. These scripts were able to extract the relevant data from the flat file database to create static html pages. After running the scripts, a page was created on the server for each protein. The html pages were then made available to the global community via the internet. Each time data were updated in the flat files, the Perl scripts had to be run again in order to reproduce the static html pages. This process of having to reproduce the static html protein pages after each database update was highly inefficient and time consuming. A more efficient approach is to produce dynamic html pages upon user request. Furthermore, the capabilities of the version 2.0 NOPdb database were limited with respect to security, ease of use, accessibility, maintainability and expandability. For example, a number of security concerns arose regarding the Perl scripts, which with limited documentation, proved very difficult to resolve.
The new version of the NOPdb3.0 (http://www.lamondlab.com/NOPdb3.0/) consists of a unique, secure, extendable content management system, holding advanced nucleolar proteomics data. The created application includes a dynamic relational database, which is kept up to date by members of the Lamond group. It also allows the query of protein data hosted within the database by external users, either using the custom built interface provided by the Lamond group, or by building custom web tools that access data via the Application Programming Interface (API). In addition to the dynamic interfaces provided by the new content management system, the data included in the nucleolar proteome are also dynamically updated with proteins identified from several different cell lines, using various instruments by members of the laboratory. From the initial 700 proteins identified in the previous iteration of the NOPdb, we have now identified over 50 000 peptides contained in over 4500 human proteins from purified nucleoli, providing significantly enhanced coverage of the nucleolar proteome.
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DATABASE ACCESS
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We have established the new version of the Nucleolar Proteome
Database (NOPdb3.0), which archives all the human nucleolar
proteins identified to date by the Lamond group and their collaborators
using MS analyses (
1–3). This current version 3.0 of the
database is available at
http://www.lamondlab.com/NOPdb3.0/ and is searchable either by protein name, protein sequence,
motif (
4–6), Gene Ontology (GO) (
7) terms or by setting
the range of the predicted isoelectric point and/or molecular
weight (
Figure 1). To date, NOPdb3.0 archives over 4500 human
nucleolar proteins verified by multiple MS analyses in different
cell lines. The NOPdb3.0 provides information on multiple parameters,
including protein name, accession number, gene symbol, gene
name, sequence, molecular weight, isoelectric point (pI), peptides
identified, experiments in which the protein was identified,
motifs and GO annotation. The previous version of the database
(
2) will still be available through our website at
http://www.lamondlab.com/NOPdb/.
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DATABASE IMPLEMENTATION
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The new NOPdb3.0 application consists of a multi-tier architecture,
where the data storage, business logic and client interface
are separate components. The data storage is implemented via
a relational mySQL database. The database is structured (
Figure 2)
to allow easy extendibility and maintenance in the future. In
order to extract useful data, the business logic employs complex
SQL queries. The purpose of the business logic layer is to act
as an interface between the client-side application and database.
The business logic and client interface can both reside on any
Apache web server capable of serving PHP classes and the client
interface, which is built in Adobe Flex. Adobe Flex was chosen
as it allows Rich Internet Applications (RIAs) to be prototyped
and developed rapidly, with the end product running across a
wide range of client browsers.
Version 3.0 of the NOPdb is an entirely new implementation using
a fully relational design with major improvements over previous
versions and additional functionality. The newly created database
holds data of higher granularity, storing data at the peptide
level as opposed to collated data on proteins. This higher granularity
also means that results from new experiments can be directly
uploaded to the database without prior processing, as the direct
output from MS-based proteomics analyses is peptide data. The
application has the ability to interpret data and therefore
aggregate it to provide metadata for proteins on a usable, graphical
interface. The structure of the application has been designed
using the model view controller design pattern (
8), thus meaning
that the functionality is separated from the overall look and
feel of the application to ensure a more customisable solution.
All communication between the database and application has been
implemented to pass through the custom made API (
9). Furthermore,
in this new version 3.0 application, the graphical user interface
to the database is able to create data pages ‘on the fly’
using the custom API rather than serving static data pages,
as in previous versions. This API not only acts as a security
blanket around the database, it also provides the ability for
users to create their own websites and/or applications that
represent the data being stored in the proteomics database.
External users can make use of the API through the REST (Representational
State Transfer) (
10) approach. Hence, external programmers can
retrieve content in XML (Extensible Markup Language) format,
from the database, by accessing well-documented Uniform Resource
Locators (URLs).
The application also facilitates mining of stored data, with data being stored in a relational structure that is well documented. Thus tools can be built to search, analyse, read and understand the data. This mining capability is evident within the application, with the database being searchable by multiple parameters, including gene names, amino acid or nucleotide sequences, sequence motifs, or by limiting the range for isoelectric points and/or molecular weights. The database is also searchable by Interpro motif numbers (database of protein families, domains and functional sites) (4–6) and by GO terms (describe gene products in terms of their associated biological processes, cellular components and molecular functions in a species-independent manner) (7). Furthermore, the NOPdb3.0 application uses the API to create dynamically generated graphs, allowing the users to visualise the data produced from experiments and enabling cross analysis between experiments.
Increased security was a core focus of this development. The application itself is designed with three levels of access, to facilitate management and to prevent unauthorised use of the system. Users are provided with different levels of access according to their needs, which are seamlessly enforced by the application. This security ensures that the data remain accurate and the quality of the data is not compromised. Furthermore, this application creates a platform for the Lamond group to share their data with the wider cell biology community.
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DATABASE CONTENT
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The database has been populated with different sets of experiments,
performed in the Lamond laboratory, that identify proteins in
purified preparations of human nucleoli. This new NOPdb3.0 now
contains over 4500 proteins identified in different human cells
lines. The increased coverage of the human nucleolus proteome
is illustrated by the fact that NOPdb3.0 now includes over 80%
of ribosomal proteins, as opposed to the
28% described in NOPdb
version 2.0. We estimate that NOPdb3.0 contains over 80% of
the main human nucleolus proteins. The proteins in the database
will be regularly updated as more experiments are performed
in the Lamond laboratory.
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FUNDING
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This work was supported by a Wellcome Trust Programme Grant
(073980/Z/03/Z) and by an interdisciplinary RASOR (Radical Solutions
for Researching the Proteome) initiative, which is supported
by the Biotechnology and Biological Sciences Research Council,
Engineering and Physical Sciences Research Council, Scottish
Higher Education Funding Council and Medical Research Council.
A.I.L. is a Wellcome Trust Principal Research Fellow. Caledonian
Research Foundation Fellowship (to F.M.B.). BBSRC PhD studentship
(to Y.A.). Funding for open access charge: Wellcome Trust.
Conflict of interest statement. None declared.
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ACKNOWLEDGEMENTS
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We would like to thank Drs Douglas Lamont and Kenneth Beattie
of the Fingerprints Proteomics Facility at the University of
Dundee (
http://proteomics.lifesci.dundee.ac.uk/) for technical
assistance.
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ABSTRACT
INTRODUCTION
