Targeted mass spectrometry: An emerging powerful approach to unblock the bottleneck in phosphoproteomics.

Following the rapid expansion of the proteomics field, the investigation of post translational modifications (PTM) has become extremely popular changing our perspective of how proteins constantly fine tune cellular functions. Reversible protein phosphorylation plays a pivotal role in virtually all biological processes in the cell and it is one the most characterized PTM up to date. During the last decade, the development of phosphoprotein/phosphopeptide enrichment strategies and mass spectrometry (MS) technology has revolutionized the field of phosphoproteomics discovering thousands of new site-specific phosphorylations and unveiling unprecedented evidence about their modulation under distinct cellular conditions. The field has expanded so rapidly that the use of traditional methods to validate and characterize the biological role of the phosphosites is not feasible any longer. Targeted MS holds great promise for becoming the method of choice to study with high precision and sensitivity already known site-specific phosphorylation events. This review summarizes the contribution of large-scale unbiased MS analyses and highlights the need of targeted MS-based approaches for follow-up investigation. Additionally, the article illustrates the biological relevance of protein phosphorylation by providing examples of disease-related phosphorylation events and emphasizes the benefits of applying targeted MS in clinics for disease diagnosis, prognosis and drug-response evaluation.

Interleukin-2 signaling pathway analysis by quantitative phosphoproteomics.

Interleukin-2 (IL-2) is major cytokine involved in T cell proliferation, differentiation and apoptosis. Association between IL-2 and its receptor (IL-2R), triggers activation of complex signaling cascade governed by tyrosine phosphorylation that culminates in transcription of genes involved in modulation of the immune response. The complete characterization of the IL-2 pathway is essential to understand how aberrant IL-2 signaling results in several diseases such as cancer or autoimmunity and also how IL-2 treatments affect cancer patients. To gain insights into the downstream machinery activated by IL-2, we aimed to define the global tyrosine-phosphoproteome of IL-2 pathway in human T cell line Kit225 using high resolution mass spectrometry combined with phosphotyrosine immunoprecipitation and SILAC. The molecular snapshot at 5min of IL-2 stimulation resulted in identification of 172 proteins among which 79 were found with increased abundance in the tyrosine-phosphorylated complexes, including several previously not reported IL-2 downstream effectors. Combinatorial site-specific phosphoproteomic analysis resulted in identification of 99 phosphorylated sites mapping to the identified proteins with increased abundance in the tyrosine-phosphorylated complexes, of which 34 were not previously described. In addition, chemical inhibition of the identified IL-2-mediated JAK, PI3K and MAPK signaling pathways, resulted in distinct alteration on the IL-2 dependent proliferation.

Computational approach for identification and characterization of GPI-anchored peptides in proteomics experiments.

Genes that encode glycosylphosphatidylinositol anchored proteins (GPI-APs) constitute an estimated 1-2% of eukaryote genomes. Current computational methods for the prediction of GPI-APs are sensitive and specific; however, the analysis of the processing site (omega- or omega-site) of GPI-APs is still challenging. Only 10% of the proteins that are annotated as GPI-APs have the omega-site experimentally verified. We describe an integrated computational and experimental proteomics approach for the identification and characterization of GPI-APs that provides the means to identify GPI-APs and the derived GPI-anchored peptides in LC-MS/MS data sets. The method takes advantage of sequence features of GPI-APs and the known core structure of the GPI-anchor. The first stage of the analysis encompasses LC-MS/MS based protein identification. The second stage involves prediction of the processing sites of the identified GPI-APs and prediction of the corresponding terminal tryptic peptides. The third stage calculates possible GPI structures on the peptides from stage two. The fourth stage calculates the scores by comparing the theoretical spectra of the predicted GPI-peptides against the observed MS/MS spectra. Automated identification of C-terminal GPI-peptides from porcine membrane dipeptidase, folate receptor and CD59 in complex LC-MS/MS data sets demonstrates the sensitivity and specificity of this integrated computational and experimental approach.

Phosphorylation of both nucleoplasmin domains is required for activation of its chromatin decondensation activity.

Nucleoplasmin (NP) is a histone chaperone involved in nucleosome assembly, chromatin decondensation at fertilization, and apoptosis. To carry out these activities NP has to interact with different types of histones, an interaction that is regulated by phosphorylation. Here we have identified a number of phosphorylated residues by mass spectrometry and generated mutants in which these amino acids are replaced by Asp to mimic the effect of phosphorylation. Our results show that, among the eight phosphoryl groups experimentally detected, four are located at the flexible N terminus, and the rest are found at the tail domain, flanking the nuclear localization signal. Phosphorylation-mimicking mutations render a recombinant protein as active in chromatin decondensation as hyperphosphorylated NP isolated from Xenopus laevis eggs. Comparison of mutants in which the core and tail domains of the protein were independently or simultaneously "activated" indicates that activation or phosphorylation of both protein domains is required for NP to efficiently extract linker-type histones from chromatin.

Fungicidal monoclonal antibody C7 binds to Candida albicans Als3.

Monoclonal antibody (MAb) C7 reacted with a >200-kDa component from the Candida albicans cell wall identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry as Als3. It also bound the recombinant N terminus of Als3. Binding of MAb C7 to Als3 may explain the biological activities exerted by the MAb on C. albicans.

Isolation and characterization of glycosylphosphatidylinositol-anchored peptides by hydrophilic interaction chromatography and MALDI tandem mass spectrometry.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are posttranslationally processed proteins that become tethered to the extracellular leaflet of the plasma membrane via a C-terminal glycan-like moiety. Since the first GPI-AP was described in the 1970s, more than 500 GPI-APs have been reported in a range of species, including plants, microbes, and mammals. GPI-APs are probably involved in cell signaling, cell recognition, and cell remodeling processes, and they may potentially serve as cell surface antigens or vaccine targets in pathogenic microorganisms or transformed mammalian cells. Due to the structural complexity and physicochemical properties of GPI-APs, their identification and structural characterization is a demanding analytical task. Here, we report a simple, fast and sensitive method for isolation and structural analysis of GPI-anchors using a combination of hydrophilic interaction liquid chromatography and matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight tandem mass spectrometry. This method allowed analysis of GPI peptides derived from low picomole levels of the porcine kidney membrane dipeptidase. Furthermore, it allowed unambiguous assignment of the omega site via amino acid sequencing of the modified peptides. GPI-anchor-specific diagnostic ions were observed by MALDI-MS/MS at m/z 162, 286, 422, and 447, corresponding to glucosamine, mannose ethanolamine phosphate, glucosamine inositol phosphate, and mannose ethanolamine phosphate glucosamine, respectively. Thus, the methodology described herein may enable sensitive and specific detection of GPI-anchored peptides in large-scale proteomic studies of plasma membrane proteins.

A monoclonal antibody directed against a Candida albicans cell wall mannoprotein exerts three anti-C. albicans activities.

Antibodies are believed to play a role in the protection against Candida albicans infections by a number of mechanisms, including the inhibition of adhesion or germ tube formation, opsonization, neutralization of virulence-related enzymes, and direct candidacidal activity. Although some of these biological activities have been demonstrated individually in monoclonal antibodies (MAbs), it is not clear if all these anti-C. albicans activities can be displayed by a single antibody. In this report, we characterized a monoclonal antibody raised against the main target of salivary secretory immunoglobulin A in the cell wall of C. albicans, which exerts three anti-C. albicans activities: (i) inhibition of adherence to HEp-2 cells, (ii) inhibition of germination, and (iii) direct candidacidal activity. MAb C7 reacted with a proteinic epitope from a mannoprotein with a molecular mass of >200 kDa predominantly expressed on the C. albicans germ tube cell wall surface as well as with a number of antigens from Candida lusitaniae, Cryptococcus neoformans, Aspergillus fumigatus, and Scedosporium prolificans. MAb C7 caused a 31.1% inhibition in the adhesion of C. albicans to HEp-2 monolayers and a 55.3% inhibition in the adhesion of C. albicans to buccal epithelial cells, produced a 38.5% decrease in the filamentation of C. albicans, and exhibited a potent fungicidal effect against C. albicans, C. lusitaniae, Cryptococcus neoformans, A. fumigatus, and S. prolificans, showing reductions in fungal growth ranging from 34.2 to 88.7%. The fungicidal activity showed by MAb C7 seems to be related to that reported by antibodies mimicking the activity of a killer toxin produced by the yeast Pichia anomala, since one of these MAbs also reacted with the C. albicans mannoprotein with a molecular mass of >200 kDa. Results presented in this study support the concept of a family of microbicidal antibodies that could be useful in the treatment of a wide range of microbial infections when used alone or in combination with current antimicrobial agents.