The analysis of phosphoproteomes by selective labelling and advanced mass spectrometric techniques.

This chapter focuses on the development of new proteomic approaches based on classical biochemical procedures coupled with new mass spectrometry methods to study the phosphorylation, the most important and abundant PTMs in modulating protein activity and propagating signals within cellular pathways and networks. These phosphoproteome studies aim at comprehensive analysis of protein phosphorylation by identification of the phosphoproteins, exact localization of phosphorylated residues, and preferably quantification of the phosphorylation. Because of low stoichiometry, heterogeneity, and low abundance, enrichment of phosphopeptides is an important step of this analysis. The first section is focused on the development of new enrichment methods coupled to mass spectrometry. Thus, improved approach, based on simple chemical manipulations and mass spectrometric procedures, for the selective analysis of phosphoserine and phosphothreonine in protein mixtures, following conversion of the peptide phosphate moiety into DTT derivatives, is described. However the major aim of this work is devoted to the use of isotopically labelled DTT, thus allowing a simple and direct quantitative MS analysis. The final part of the work is focused on the development of a strategy to study phosphorylation without preliminary enrichment but using the high performance of a novel hybrid mass spectrometer linear ion trap.

Synthesis and proteomic activity evaluation of a new isotope-coded affinity tagging (ICAT) reagent.

During recent years, quantitative proteome profiling has taken advantage of incorporating the traditional stable isotope dilution analysis into global scale or discovery-based proteomic experiments that use mass spectrometers as detectors to allow the pairwise study of differently expressed proteins. Quantitative protein analysis by means of the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS) enables the pairwise comparison of protein expression levels in biological samples. Herein, a modified ICAT reagent, named BAA-ICAT (beta-alanine-arm-ICAT) in which the polyether linker is replaced by a more water-soluble polyamide one, was investigated.

Selective detection and identification of phosphopeptides by dansyl MS/MS/MS fragmentation.

Protein phosphorylation regulates many cellular processes and pathways, such as cell cycle progression, signal transduction cascades and gene expression. Selective detection of phosphopeptides from proteolytic digests is a challenging and highly relevant task in many proteomics applications. Often phosphopeptides are present in small amounts and need selective isolation or enrichment before identification. Here we report a novel approach to label selectively phospho-Ser/-Thr residues by exploiting the features of a novel linear ion trap mass spectrometer. Using dansyl labelling and MS3 fragmentation, we developed a method useful for the large-scale proteomic profiling of phosphorylation sites. The new residues in the sequence were stable and easily identifiable under general conditions for tandem mass spectrometric sequencing.

Tubulin nitration in human gliomas.

Immunohistochemical and biochemical investigations showed that significant protein nitration occurs in human gliomas, especially in grade IV glioblastomas at the level of astrocytes and oligodendrocytes and neurones. Enhanced alpha-tubulin immunoreactivity was co-present in the same elements in the glioblastomas. Proteomic methodologies were employed to identify a nitrated protein band at 55 kDa as alpha-tubulin. Peptide mass fingerprinting procedures demonstrated that tubulin is nitrated at Tyr224 in grade IV tumour samples but is unmodified in grade I samples and in non-cancerous brain tissue. These results provide the first characterisation of endogenously nitrated tubulin from human tumour samples.

Monitoring food quality by microfluidic electrophoresis, gas chromatography, and mass spectrometry techniques: effects of aquaculture on the sea bass (Dicentrarchus labrax).

Monitoring food quality is a critical task for analytical chemistry and an important way to preserve human health. Fish is a valuable source of highly digestible proteins and contains large amounts of polyunsaturated fatty acids and fat-soluble vitamins. Since the world's wild fish stocks are limited, farmed fish is nowadays proposed as an alternative to consumers. It is now emerging that the fish muscle protein content is assuming great importance from an aquaculture perspective. Many data have been collected on the physiology and biochemistry of fish muscle, but few proteomic studies are available on farmed fish. Application of proteomics to aquaculture may play a key role in the development of new farming strategies. In this paper, a proteomic approach based on SDS-PAGE separation of proteins, in situ protein hydrolysis, de novo sequencing of peptides by MALDI and ESI MS(2), protein identification, and relative quantitation of protein by denaturing capillary electrophoresis was coupled with the determination of fatty acids and metal ions content by GM-MS and ICPMS in farmed and wild sea bass filet. Our results show that aquaculture could induce significant chemical and biochemical differences in fish muscle that may have an impact on food quality.

Direct interactions among Ret, GDNF and GFRalpha1 molecules reveal new insights into the assembly of a functional three-protein complex.

The glial-cell-line-derived neurotrophic factor (GDNF) ligand activates the Ret receptor through the assembly of a multiprotein complex, including the GDNF family receptor alpha1 (GFRalpha1) molecule. Given the neuroprotective role of GDNF, there is an obvious need to precisely identify the structural regions engaged in direct interactions between the three molecules. Here, we combined a functional approach for Ret activity (in PC12 cells) to cross-linking experiments followed by MS-MALDI to study the interactions among the purified extracellular region of the human Ret, GDNF and GFRalpha1 molecules. This procedure allowed us to identify distinct regions of Ret that are physically engaged in the interaction with GDNF and GFRalpha1. The lack of these regions in a recombinant Ret form results in the failure of both structural and functional binding of Ret to GFRalpha1/GDNF complex. Furthermore, a model for the assembly of a transducing-competent Ret complex is suggested.

Ribosomal protein L7a binds RNA through two distinct RNA-binding domains.

The human ribosomal protein L7a is a component of the major ribosomal subunit. We previously identified three nuclear-localization-competent domains within L7a, and demonstrated that the domain defined by aa (amino acids) 52-100 is necessary, although not sufficient, to target the L7a protein to the nucleoli. We now demonstrate that L7a interacts in vitro with a presumably G-rich RNA structure, which has yet to be defined. We also demonstrate that the L7a protein contains two RNA-binding domains: one encompassing aa 52-100 (RNAB1) and the other encompassing aa 101-161 (RNAB2). RNAB1 does not contain any known nucleic-acid-binding motif, and may thus represent a new class of such motifs. On the other hand, a specific region of RNAB2 is highly conserved in several other protein components of the ribonucleoprotein complex. We have investigated the topology of the L7a-RNA complex using a recombinant form of the protein domain that encompasses residues 101-161 and a 30mer poly(G) oligonucleotide. Limited proteolysis and cross-linking experiments, and mass spectral analyses of the recombinant protein domain and its complex with poly(G) revealed the RNA-binding region.