In-depth profiling of the peripheral blood mononuclear cells proteome for clinical blood proteomics.

Peripheral blood mononuclear cells (PBMCs) are an easy accessible cellular part of the blood organ and, along with platelets, represent the only site of active gene expression in blood. These cells undergo immunophenotypic changes in various diseases and represent a peripheral source of monitoring gene expression and posttranslational modifications relevant to many diseases. Little is known about the source of many blood proteins and we hypothesise that release from PBMCs through active and passive mechanisms may account for a substantial part of the plasma proteome.

TMT labelling for the quantitative analysis of adaptive responses in the meningococcal proteome.

In addition to standard gel-based proteomic approaches, gel-free approaches using isobaric label reagents, such as Tandem Mass Tags (TMT), provide a straightforward method for studying adaptations in microbial proteomes to changing environmental conditions. This approach does not have the known difficulties of 2-D gel electrophoresis with proteins of extreme biochemical properties. The workflow described here was designed to study adaptive responses in bacteria and has been applied to study the response of meningococci to iron limitation.

Candidate verification of iron-regulated Neisseria meningitidis proteins using isotopic versions of tandem mass tags (TMT) and single reaction monitoring.

Tandem Mass Tags (TMT) are suited to both global and targeted quantitation approaches of proteins and peptides. Different versions of these tags allow for the generation of both isobaric and isotopic sets of reagents sharing the same common structure. This feature allows for a straightforward transfer of data obtained during discovery studies into targeted investigations.

Identification of proteins of Neisseria meningitidis induced under iron-limiting conditions using the isobaric tandem mass tag (TMT) labeling approach.

Isobaric labeling reagents such as Tandem Mass Tags (TMT(R)) enable the genome-wide quantification of protein expression levels under different conditions using a gel-free MS/MS-based approach. Here, we applied a TMTduplex approach with two isobaric tags to study the response of the human pathogen Neisseria meningitidis to deprivation of iron, a condition met in the human body. In total, 609 proteins were identified in samples of three independent growth experiments, in which we compared cultures grown in the presence and absence of iron.

Comparison of the nuclear proteomes of mammary epithelial cells at different stages of functional differentiation.

The progression of stem cells to proliferating progenitor cells and finally to a quiescent differentiated state is a hallmark of organ development. This process proceeds through distinct steps and is regulated through cell-cell interactions and by systemically and locally acting factors. We have established a cell culture system which recapitulates features of mammary gland development in vitro and allows the comparison of three characteristic differentiation stages.

Protein sequence tags: a novel solution for comparative proteomics.

Comparative proteome profiling using stable isotope peptide labelling and mass spectrometry has emerged as a promising strategy. Here, we show the broad potential of our proprietary protein sequence tag (PST) technology. A special feature of PST is its ability to detect a wide variety of proteins including the pharmaceutically relevant membrane and nuclear proteins.

Characterization of low abundant membrane proteins using the protein sequence tag technology.

About 25% of open reading frames in fully sequenced genomes are estimated to encode transmembrane proteins that represent valuable targets for drugs. However, the global analysis of membrane proteins has been proven to be problematic, e.g.

Isolation of N-terminal protein sequence tags from cyanogen bromide cleaved proteins as a novel approach to investigate hydrophobic proteins.

A novel method for the isolation of protein sequence tags to identify proteins in a complex mixture of hydrophobic proteins is described. The PST (Protein Sequence Tag) technology deals with the isolation and MS/MS based identification of one N-terminal peptide from each polypeptide fragment generated by cyanogen bromide cleavage of a mixture of proteins. PST sampling takes place after sub-cellular fractionation of a complex protein mixture to give enrichment of mitochondrial proteins.

Comparative proteomic analysis of proliferating and functionally differentiated mammary epithelial cells.

Proliferation and differentiation of mammary epithelial cells are governed by hormonal stimuli, cell-cell, and cell-matrix interactions. Terminal differentiation of mammary epithelial cells depends upon the action of the lactogenic hormones, insulin, glucocorticoids, and prolactin that enable them to synthesize and secrete milk proteins. These differentiated cells are polarized and carry out vectorial transport of milk constituents across the apical plasma membrane.

Biogenesis of yeast mitochondrial cytochrome c: a unique relationship to the TOM machinery.

The import of cytochrome c into the mitochondrial intermembrane space is not understood at a mechanistic level. While the precursor apocytochrome c can insert into protein-free lipid bilayers, the purified translocase of the outer membrane (TOM) complex supports the translocation of apocytochrome c into proteoliposomes. We report an in organello analysis of cytochrome c import into yeast mitochondria from wild-type cells and different mutants cells, each defective in one of the seven Tom proteins.

Protein translocase of the outer mitochondrial membrane: role of import receptors in the structural organization of the TOM complex.

The mitochondrial outer membrane contains a multi-subunit machinery responsible for the specific recognition and translocation of precursor proteins. This translocase of the outer membrane (TOM) consists of three receptor proteins, Tom20, Tom22 and Tom70, the channel protein Tom40, and several small Tom proteins. Single-particle electron microscopy analysis of the Neurospora TOM complex has led to different views with two or three stain-filled centers resembling channels.