Current Proteomics

ISSN: 1570-1646

Current Proteomics
Volume 4 Number 4, December 2007

Contents


Possibilities of Two-Dimensional Gel Electrophoresis in the Understanding of Human Disease Pp. 187-197
J. Bermúdez-Crespo and J.L. López
[Abstract]


The “Invisible Proteome”: How to Capture the Low Abundance Proteins Via Combinatorial Ligand Libraries Pp. 198-208
E. Boschetti, B. Monsarrat and P.G. Righetti
[Abstract]


Neuroproteomics and the Detection of Regulatory Phosphosites Pp. 209-222
R.Y. Tweedie-Cullen, B. Wollscheid, M. Livingstone-Zatchej and I.M. Mansuy
[Abstract]


Computational Methods and Algorithms for Mass Spectrometry Based Differential Proteomics Pp. 223-234
S. Mavroudi, S. Papadimitriou, S. Kossida, S.D. Likothanassis and A. Vlahou
[Abstract]


Relevance of Protein Isoforms in Proteomic Studies Pp. 235-252
A.M. Rodríguez-Piñeiro, P. Álvarez-Chaver, V.S. Martínez Zorzano, F.J. Rodríguez-Berrocal and M.P. de la Cadena
[Abstract]



Abstracts


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Possibilities of Two-Dimensional Gel Electrophoresis in the Understanding of Human Disease
J. Bermúdez-Crespo and J.L. López

Because of the multifactorial nature of many diseases, two-dimensional electrophoresis is a basic proteomics tool for its ability of simultaneously detecting post-and co-translational modifications, which cannot be predicted from genome sequences.

This review describes the central role of proteomics tool, two-dimensional electrophoresis for clinical biomarker discovery, the identification of prognostic and diagnostic markers, their use in monitoring the effects of drug treatments and eventually finding more efficient and safer therapeutics for a wide range of pathologies.


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The “Invisible Proteome”: How to Capture the Low Abundance Proteins Via Combinatorial Ligand Libraries
E. Boschetti, B. Monsarrat and P.G. Righetti

Despite recent advances in pre-fractionation procedures and depletion methods via immuno-subtraction protocols of the most abundant species, the “low-abundance” proteome remains largely undetected. We report here a novel technology, called ProteoMiner, for bringing to the limelight this vast pool of low-abundance species, which could constitute >50% of any proteome in any living organism. It consists of a combinatorial ligand library, composed of millions of diverse hexapeptide baits, able to capture and concentrate the “low-abundance” proteome, while drastically cutting the concentration of the most abundant compounds. Since no depletion of any species is contemplated by this methodology, but a drastic reduction of the sample dynamic range, the noxious phenomenon of co-depletion (especially troublesome in affinity depletion methods) does not occur. In addition to previously reported data on analysis of human sera and urines, we describe here novel applications for the detection of the low-abundance proteome in human blood cells, such as the red blood cells (RBC) and platelets and in biological fluids, such as cerebrospinal fluid. In particular, in the case of RBC, where hemoglobin alone constitutes ca. 98% of the total cytoplasmic proteome, the ProteoMiner technology allowed the detection of >1500 proteins in the remaining 2% low-abundance cytoplasmic proteome, most of them not previously reported even in the most advanced investigations. The merits and limits of ProteMiner are discussed and evaluated.


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Neuroproteomics and the Detection of Regulatory Phosphosites
R.Y. Tweedie-Cullen, B. Wollscheid, M. Livingstone-Zatchej and I.M. Mansuy

Protein phosphorylation is a key post-translational modification that controls intracellular signalling in virtually all cell types. In the nervous system, it contributes to the regulation of neuronal signalling and control processes underlying synaptic plasticity and cognitive functions. However, despite its importance, knowledge about phosphoproteins and their phosphosites in the brain remains limited. A pre-requisite for unravelling brain biology and function at the molecular level, are the qualitative and quantitative analyses of protein phosphorylation and its dynamics. These analyses of the phosphoproteome require novel methodologies in addition to traditional biochemical methods. Current phosphoproteomic workflows have reached a level of maturity, which allow for their use in combination with molecular approaches, and their application to the study of higher order brain function and cognitive processes. Neuroproteomics is emerging as an essential new sub-field of the neurosciences. This review focuses on the recent advances in the application of neuroproteomics to the phosphoproteome and discusses the challenges to come.


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Computational Methods and Algorithms for Mass Spectrometry Based Differential Proteomics
S. Mavroudi, S. Papadimitriou, S. Kossida, S.D. Likothanassis and A. Vlahou

As most high-throughput data, mass spec proteomics data are complex, noisy and incomplete. Additionally, in settings addressing questions about differential expression of proteins the data are usually represented by relatively few samples and a very large number of predictor variables, i.e., m/z peaks. These characteristics pose a significant challenge for most analysis methods. In addition, the preprocessing of the data remains an active research area having a great impact on the subsequent analysis steps.

A wide range of algorithms have been proposed for both the pre-processing and the higher leve l analysis of proteomics data. They range from classical approaches to second generation algorithms, which aim at tackling some of the limitations of earlier methods. Many of the proposed algorithms have been reported to produce encouraging results. However, no single algorithm has emerged as a method of choice.

This work provides a critical review of the recent approaches for pre-processing and higher level analysis of proteomics data. Also their strengths and limitations are evaluated. Emphasis is given on describing the most common and serious mistakes recorded in published differential proteomics studies. Moreover, the review provides guidance for choosing and correctly applying the appropriate algorithms according to our experience. Also hints for the design of novel algorithms, which will more effectively handle the specific characteristics and constrains of differential proteomics data are discussed.


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Relevance of Protein Isoforms in Proteomic Studies
A.M. Rodríguez-Piñeiro, P. Álvarez-Chaver, V.S. Martínez Zorzano, F.J. Rodríguez-Berrocal and M.P. de la Cadena

The development of proteomics has generated considerable interest in the search of new biomarkers since proteins reflect biological conditions more directly than nucleic acids. However, despite the large number of laboratories reporting exciting and successful studies within this frame, we have not witnessed its clinical application yet. One of the reasons for this failure is the difficult validation of the results extracted from differential protein expression studies, mainly because the numerous pre- and post-translational events lead to the appearance of the so-called isoforms, which the immunochemical methods employed fail to distinguish due to a lack of isoform-specific antibodies.

Though the term “isoform” does not exist as such according to the IUPAC, with the development of proteomics this de-nomination is used to refer to various forms of a protein which charge or mass properties produce different mobility in two-dimensional gels, irrespectively of their genetic origin. In this review, we address this issue and consider the different definitions of “isoforms”; we also explain the origins of the protein diversity, from the early mechanisms of RNA editing and alternative splicing to the different types of post-translational modifications. From the research point of view, we address the utility of the proteomic methods that allow isoform detection and distinction, as well as the issue of isoform annotation in databases. From an applied point of view, we consider the problem isoforms involve in the clinical practice, together with their relevance in the disease biomarker field and their role during validation. Lastly, we provide some examples of well-known proteins for which isoforms have been reported in literature.