CompOmics

An analysis of the resolution limitations of peptide identification algorithms

martlenn — Tue, 18 Oct 2011 09:35:11 +0000

Proteome identification using peptide-centric proteomics techniques is a routinely used analysis technique. One of the most powerful and popular methods for the identification of peptides from MS/MS spectra is protein database matching using search engines. Significance thresholding through false discovery rate (FDR) estimation by target/decoy searches is used to ensure the retention of predominantly confident assignments of MS/MS spectra to peptides. However, shortcomings have become apparent when such decoy searches are used to estimate the FDR. In order to study these shortcomings, we here introduce a novel kind of decoy database that contains isobaric mutated versions of the peptides that were identified in the original search. Because of the supervised way in which the entrapment sequences are generated, we call this a directed decoy database. Since the peptides found in our directed decoy database are thus specifically designed to look quite similar to the forward identifications, the limitations of the existing search algorithms in making correct calls in such strongly confusing situations can be analyzed. Interestingly, for the vast majority of confidently identified peptide identifications, a directed decoy peptide-to-spectrum match can be found that has a better or equal match score than the forward match score, highlighting an important issue in the interpretation of peptide identifications in present-day high-throughput proteomics.

Colaert N, Degroeve S, Helsens K, Martens L. An analysis of the resolution limitations of peptide identification algorithms. J Proteome Res. 2011 Oct 13. [Epub ahead of print]

pubmed: http://www.ncbi.nlm.nih.gov/pubmed/21995378

Filed under: 2011, Publications

jTraML: An Open Source Java API for TraML, the PSI Standard for Sharing SRM Transitions

martlenn — Tue, 18 Oct 2011 09:34:05 +0000

We here present jTraML, a Java API for the Proteomics Standards Initiative TraML data standard. The library provides fully functional classes for all elements specified in the TraML XSD document, as well as convenient methods to construct controlled vocabulary-based instances required to define SRM transitions. The use of jTraML is demonstrated via a two-way conversion tool between TraML documents and vendor specific files, facilitating the adoption process of this new community standard. The library is released as open source under the permissive Apache2 license and can be downloaded from http://jtraml.googlecode.com . TraML files can also be converted online at http://iomics.ugent.be/jtraml .

Helsens K, Brusniak MY, Deutsch E, Moritz RL, Martens L. jTraML: An Open Source Java API for TraML, the PSI Standard for Sharing SRM Transitions. J Proteome Res. 2011 Oct 13. [Epub ahead of print]

pubmed: http://www.ncbi.nlm.nih.gov/pubmed/21967198

Filed under: 2011, Publications

Bioinformatics challenges in the proteomic analysis of human plasma

martlenn — Tue, 18 Oct 2011 09:28:58 +0000

Mass spectrometry has become the method of choice for studying proteins in complex mixtures in a qualitative and quantitative fashion. The application of mass spectrometry-based proteomics analyses on plasma has correspondingly been established as an important method for disease-associated biomarker discovery and validation. Yet despite being a readily available human sample, plasma poses several important challenges to the proteomics researcher. With a focus on bioinformatics aspects, this chapter will discuss the problems involved in analyzing plasma proteomics data, along with the scope of solutions available through specialised tools and sophisticated analysis methods.

Foster JM, Martens L. Bioinformatics challenges in the proteomic analysis of human plasma. Methods Mol Biol. 2011;728:333-47.

pubmed: http://www.ncbi.nlm.nih.gov/pubmed/21468959

Filed under: 2011, Publications

Bioinformatics challenges in mass spectrometry-driven proteomics

martlenn — Tue, 18 Oct 2011 09:20:17 +0000

Mass spectrometry-based proteomics has become an essential part of the analytical toolbox of the life sciences. With the ability to identify and quantify hundreds to thousands of proteins in high throughput, the field has contributed its fair share to the data avalanche coming from the so-called omics fields. As a result, the challenges involved in processing and managing this flood of data have grown as well. This chapter will point out and discuss these challenges, starting from the processing of raw mass spectrometry data into peaks, over the identification of peptides and proteins, to the quantification of the identified molecules. Finally, the informatics aspects of the nascent field of targeted proteomics are outlined as well.

Martens L. Bioinformatics challenges in mass spectrometry-driven proteomics. Methods Mol Biol. 2011;753:359-71.

pubmed: http://www.ncbi.nlm.nih.gov/pubmed/21604135

Filed under: 2011, Publications

thermo-msf-parser: an open source Java library to parse and visualize Thermo Proteome Discoverer msf files

kennyhelsens — Fri, 08 Jul 2011 07:49:53 +0000

The Thermo Proteome Discoverer program integrates both peptide identification and quantification into a single workflow for peptide-centric proteomics. Furthermore, its close integration with Thermo mass spectrometers has made it increasingly popular in the field. Here, we present a Java library to parse the msf files that constitute the output of Proteome Discoverer. The parser is also implemented as a graphical user interface allowing convenient access to the information found in the msf files, and in Rover, a program to analyse and validate quantitative proteomics information. All code, binaries and documentation is freely available at http://thermo-msf-parser.googlecode.com.
Colaert N, Barsnes H, Vaudel M, Helsens K, Timmerman E, Sickmann A, Gevaert K, Martens L. thermo-msf-parser: an open source Java library to parse and visualize Thermo Proteome Discoverer msf files. J Proteome Res. 2011 Jun 30. [Epub ahead of print]

pubmed: http://www.ncbi.nlm.nih.gov/pubmed/21714566

Filed under: 2011, Publications

PhD Niklaas Colaert

niklaascolaert — Mon, 06 Jun 2011 12:27:16 +0000

On Friday 13th of May 2011, Niklaas Colaert has successfully defended his doctoral thesis.
If interested, please find an electronic copy of the thesis below!

Thesis_Colaert: download

Filed under: News

Bioinformatics analysis of a Saccharomyces cerevisiae N-terminal proteome provides evidence of alternative translation initiation and post-translational N-terminal acetylation.

niklaascolaert — Mon, 06 Jun 2011 12:13:22 +0000

Initiation of protein translation is a well studied fundamental process, albeit high-throughput and more comprehensive determination of the exact translation initiation sites (TIS) was only recently made possible following the introduction of positional proteomics techniques that target protein N-termini. Precise translation initiation is of crucial importance, as truncated or extended proteins might fold, function and locate erroneously. Still, as already shown for some proteins, alternative translation initiation can also serve as a regulatory mechanism. By applying N-terminal COFRADIC (combined fractional diagonal chromatography), we here isolated N-terminal peptides of a S. cerevisiae proteome and analyzed both annotated and alternative TIS. We analyzed this N-terminome of S. cerevisiae which resulted in the identification of 650 unique N-terminal peptides corresponding to database annotated TIS. Furthermore, 56 unique Nα-acetylated peptides were identified that suggest alternative TIS (MS/MS-based), while MS-based evidence of Nα-acetylation led to an additional 33 such peptides. To improve the overall sensitivity of the analysis, we also included the 5′ UTR (untranslated region) in-frame translations together with the yeast protein sequences in UniProtKB/Swiss-Prot. To ensure the quality of the individual peptide identifications, peptide-to-spectrum-matches were only accepted at a 99% probability threshold, and were subsequently analysed in detail by the Peptizer tool to automatically ascertain their compliance with several expert criteria. Furthermore, we have also identified 60 MS/MS-based and 117 MS-based Nα-acetylated peptides that point to Nα-acetylation as a post-translational modification since these peptides did not start, nor were preceded (in their corresponding protein sequence) by a methionine residue. Next, we evaluated consensus sequence features of nucleic acids and amino acids across each of these groups of peptides and evaluated the results in the context of publicly available data. Taken together, we present a list of 706 annotated and alternative TIS for yeast proteins and found that under normal growth conditions, alternative TIS might (co-)occur in S. cerevisiae in roughly one tenth of all proteins. Furthermore, we found that the nucleic acid and amino acid features proximate to these alternative TIS favour either guanine or adenine nucleotides following the start codon, or acidic amino acids following the initiator methionine. Finally, we also observed an unexpected high number of Nα-acetylated peptides that could not be related to TIS and therefore suggest events of post-translational Nα-acetylation.

Helsens K, Van Damme P, Degroeve S, Martens L, Arnesen T, Vandekerckhove J, Gevaert K. Bioinformatics analysis of a Saccharomyces cerevisiae N-terminal proteome provides evidence of alternative translation initiation and post-translational N-terminal acetylation. J Proteome Res. 2011 May 30. [Epub ahead of print]

pubmed: http://www.ncbi.nlm.nih.gov/pubmed/21619078

Filed under: 2011, Publications

RIBAR and xRIBAR: Methods for Reproducible Relative MS/MS-based Label-Free Protein Quantification.

niklaascolaert — Wed, 25 May 2011 11:21:19 +0000

Mass spectrometry-driven proteomics is increasingly relying on quantitative analyses for biological discoveries. As a result, different methods and algorithms have been developed to perform relative or absolute quantification based on mass spectrometry data. One of the most popular quantification methods are the so-called label-free approaches, which require no special sample processing, and can even be applied retroactively to existing data sets. Of these label-free methods, the MS/MS-based approaches are most often applied, mainly because of their inherent simplicity as compared to MS-based methods. The main application of these approaches is the determination of relative protein amounts between different samples, expressed as protein ratios. However, as we demonstrate here, there are some issues with the reproducibility across replicates of these protein ratio sets obtained from the various MS/MS-based label-free methods, indicating that the existing methods are not optimally robust. We therefore present two new methods (called RIBAR and xRIBAR) that use the available MS/MS data more effectively, achieving increased robustness. Both the accuracy and the precision of our novel methods are analyzed and compared to the existing methods to illustrate the increased robustness of our new methods over existing ones.

Colaert N, Gevaert K, Martens L. RIBAR and xRIBAR: Methods for Reproducible Relative MS/MS-based Label-Free Protein Quantification. J Proteome Res. 2011 May 23. [Epub ahead of print]

pubmed: http://www.ncbi.nlm.nih.gov/pubmed/21604685

Filed under: 2011, Publications

Mass spectrometry-driven proteomics: an introduction.

niklaascolaert — Tue, 24 May 2011 07:28:50 +0000

Proteins are reckoned to be the key actors in a living organism. By studying proteins, one engages into deciphering a complex series of events occurring during a protein’s life span. This starts at the creation of a protein, which is tightly controlled on both a transcriptional (Williams and Tyler, 2007, Curr Opin Genet Dev 17, 88-93) and a translational level (Van Der Kelen et al., 2009, Crit Rev Biochem Mol Biol 44, 143-168). During translation, a primary strand of amino acids undergoes a complex folding process in order to obtain a native three-dimensional protein structure (Gross et al., 2003, Cell 115, 739-750). Proteins take on a plethora of functions, such as complex formation, receptor activity, and signal transduction, which ultimately adds up to a cellular phenotype. Consequently, protein analysis is of major interest in molecular biology and involves annotating their presence and localization, as well as their modification state and biochemical context. To accomplish this, many methods have been developed over the last decades, and their general principles and important recent advances in large-scale protein analysis or proteomics are discussed in this review.

Helsens K, Martens L, Vandekerckhove J, Gevaert K. Mass spectrometry-driven proteomics: an introduction. Methods Mol Biol. 2011;753:1-27.

pubmed: http://www.ncbi.nlm.nih.gov/pubmed/21604112

Filed under: 2011, Publications

A case study on the comparison of different software tools for automated quantification of peptides.

niklaascolaert — Tue, 24 May 2011 07:25:10 +0000

MS-driven proteomics has evolved over the past two decades to a high tech and high impact research field. Two distinct factors clearly influenced its expansion: the rapid growth of an arsenal of instrument and proteomic techniques that led to an explosion of high quality data and the development of software tools to analyze and interpret these data which boosted the number of scientific discoveries. In analogy with the benchmarking of new instruments and proteomic techniques, such software tools must be thoroughly tested and analyzed. Recently, new tools were developed for automatic peptide quantification in quantitative proteomic experiments. Here we present a case study where the most recent and frequently used tools are analyzed and compared.

Colaert N, Vandekerckhove J, Martens L, Gevaert K. A case study on the comparison of different software tools for automated quantification of peptides. Methods Mol Biol. 2011;753:373-98.

pubmed: http://www.ncbi.nlm.nih.gov/pubmed/21604136

Filed under: 2011, Publications