Quantitative proteomics using uniform 15N-labeling, mascot and the trans-proteomic pipeline

Stable isotope labeling combined with MS is a powerful method for measuring relative protein abundances, for instance, by differential metabolic labeling of some or all amino acids with 14N and 15N in cell culture or hydroponic media. These and most other types of quantitative proteomics experiments using high-throughput technologies, such as LC-MS/MS, generate large amounts of raw MS data. This data needs to be processed efficiently and automatically, from the mass spectrometer to statistically evaluated protein identifications and abundance ratios. This paper describes in detail an approach to the automated analysis of uniformly 14N/15N-labeled proteins using MASCOT peptide identification in conjunction with the trans-proteomic pipeline (TPP) and a few scripts to integrate the analysis workflow. Two large proteomic datasets from uniformly labeled Arabidopsis thaliana were used to illustrate the analysis pipeline. The pipeline can be fully automated and uses only common or freely available software.

Quantitative proteomics using uniform N-15-labeling, MASCOT, and the trans-proteomic pipeline

Stable isotope labeling combined with MS is a powerful method for measuring relative protein abundances, for instance, by differential metabolic labeling of some or all amino acids with N-14 and N-15 in cell culture or hydroponic media. These and most other types of quantitative proteomics experiments using high-throughput technologies, such as LC-MS/MS, generate large amounts of raw MS data. This data needs to be processed efficiently and automatically, from the mass spectrometer to statistically evaluated protein identifications and abundance ratios. This paper describes in detail an approach to the automated analysis of Uniformly N-14/N-15-labeled proteins using MASCOT peptide identification in conjunction with the trans-proteomic pipeline (TPP) and a few scripts to integrate the analysis workflow. Two large proteomic datasets from uniformly labeled Arabidopsis thaliana were used to illustrate the analysis pipeline. The pipeline can be fully automated and uses only common or freely available software.

Multiplexed quantitative proteomics using differential metabolic 15N-labeling

There are several advantages of using metabolic labeling in quantitative proteomics. The early pooling of samples compared to post-labeling methods eliminates errors from different sample processing, protein extraction and enzymatic digestion. Metabolic labeling is also highly efficient and relatively inexpensive compared to commercial labeling reagents. However, methods for multiplexed quantitation in the MS-domain (or ‘non-isobaric’ methods), suffer from signal dilution at higher degrees of multiplexing, as the MS/MS signal for peptide identification is lower given the same amount of peptide loaded onto the column or injected into the mass spectrometer. This may partly be overcome by mixing the samples at non-uniform ratios, for instance by increasing the fraction of unlabeled proteins. We have developed an algorithm for arbitrary degrees of nonisobaric multiplexing for relative protein abundance measurements. We have used metabolic labeling with different levels of 15N, but the algorithm is in principle applicable to any isotope or combination of isotopes. Ion trap mass spectrometers are fast and suitable for LC-MS/MS and peptide identification. However, they cannot resolve overlapping isotopic envelopes from different peptides, which makes them less suitable for MS-based quantitation. Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry is less suitable for LC-MS/MS, but provides the resolving power required to resolve overlapping isotopic envelopes. We therefore combined ion trap LC-MS/MS for peptide identification with FTICR LC-MS for quantitation using chromatographic alignment. We applied the method in a heat shock study in a plant model system (A. thaliana) and compared the results with gene expression data from similar experiments in literature.

A well-characterised peak identification list of MALDI MS profile peaks for human blood serum

MALDI MS profiling, using easily available body fluids such as blood serum, has attracted considerable interest for its potential in clinical applications. Despite the numerous reports on MALDI MS profiling of human serum, there is only scarce information on the identity of the species making up these profiles, particularly in the mass range of larger peptides. Here, we provide a list of more than 90 entries of MALDI MS profile peak identities up to 10 kDa obtained from human blood serum. Various modifications such as phosphorylation were detected among the peptide identifications. The overlap with the few other MALDI MS peak lists published so far was found to be limited and hence our list significantly extends the number of identified peaks commonly found in MALDI MS profiling of human blood serum.

Combinatorial peptide ligand libraries and plant proteomics: a winning strategy at a price

The mechanism of action and properties of a solid-phase ligand library made of hexapeptides (combinatorial peptide ligand libraries or CPLL), for capturing the "hidden proteome", i.e. the low- and very low-abundance proteins constituting the vast majority of species in any proteome, as applied to plant tissues, are reviewed here. Plant tissues are notoriously recalcitrant to protein extraction and to proteome analysis. Firstly, rigid plant cell walls need to be mechanically disrupted to release the cell content and, in addition to their poor protein yield, plant tissues are rich in proteases and oxidative enzymes, contain phenolic compounds, starches, oils, pigments and secondary metabolites that massively contaminate protein extracts. In addition, complex matrices of polysaccharides, including large amount of anionic pectins, are present. All these species compete with the binding of proteins to the CPLL beads, impeding proper capture and identification / detection of low-abundance species. When properly pre-treated, plant tissue extracts are amenable to capture by the CPLL beads revealing thus many new species among them low-abundance proteins. Examples are given on the treatment of leaf proteins, of corn seed extracts and of exudate proteins (latex from Hevea brasiliensis). In all cases, the detection of unique gene products via CPLL capture is at least twice that of control, untreated sample.

Identification of a novel mammalian post-translational modification, phosphocholine, on placental secretory polypeptides

Placental neurokinin B appears to be post-translationally modified by phosphocholine (PC) attached to the aspartyl side chain at residue 4 of the mature peptide. Corticotrophin releasing factor (CRF) was found to be expressed by the rat placenta with the main secreted forms being phosphocholinated proCRF+/- one or two polysaccharide moieties. A combination of high-pressure liquid chromatography (HPLC) and two-site immunometric analysis suggested that PC was also attached to the placental precursors of adrenocorticotrophin, hemokinin, activin and follistatin. However, the fully processed forms of rat placental activin and CRF were free of PC. Formerly, the parasitic filarial nematodes have used PC as a post-translational modification, attached via the polysaccharicle moiety of certain secretory glycoproteins to attenuate the host immune system allowing parasite survival, but it is the PC group itself which endows the carrier with the biological activity. The fact that treatment of proCRF peptides with phospholipase C but not endoglycosidase destroyed PC immunoreactivity suggested a simpler mode of attachment of PC to placental peptides than that used by nematodes. Thus, it is possible that by analogy the placenta uses its secreted phosphocholinated hormones to modulate the mother's immune system and help protect the placenta from rejection.

Retention time prediction and protein identification

Proteins are commonly identified through enzymatic digestion and generation of short sequence tags or fingerprints of peptide masses by mass spectrometry. Separation methods, such as liquid chromatography and electrophoresis, are often used to fractionate complex protein or peptide mixtures and these separations also provide information on the different species, such as molecular weight and isoelectric point from electrophoresis and hydrophobicity in reversed-phase chromatography. These are also properties that can be predicted from amino acid sequences derived from genomic sequences and used in protein identification. This chapter reviews recently introduced methods based on retention time prediction to extract information from chromatographic separations and the applications to protein identification in organisms with small and large genomes. Novel data on retention time prediction of posttranslationally modified peptides is also presented.

Identification of a serine protease involved with the regulation of adrenal growth

The adrenal cortex is a dynamic organ in which the cells of the outer cortex continually divide. It is well known that this cellular proliferation is dependent on constant stimulation from peptides derived from the ACTH precursor pro-opiomelanocortin (POMC) because disruption of pituitary corticotroph function results in rapid atrophy of the gland. Previous results from our laboratory have suggested that the adrenal mitogen is a fragment derived from the N-terminal of POMC not containing the gamma-MSH sequence. Because such a peptide is not generated during processing of POMC in the pituitary, we proposed that the mitogen is generated from circulating pro-gamma-MSH by an adrenal protease. Using degenerate oligonucleotides, we identified a secreted serine protease expressed by the adrenal gland that we named adrenal secretory protease (ASP). In the adrenal cortex, expression of ASP is limited to the outer zona glomerulosa/fasciculata, the region where cortical cells are believed to be derived, and is significantly up-regulated during compensatory growth. Y1 adrenocortical cells transfected with a vector expressing an antisense RNA (and thus having reduced levels of endogenous ASP) were found to grow slower than sense controls while also losing their ability to utilize exogenous pro-gamma-MSH in the media supporting a role for ASP in adrenal growth. Digestion of an N-POMC peptide substrate encompassing the residues around the dibasic cleavage site at positions 49/50 with affinity-purified ASP showed cleavage not to occur at the dibasic site but two residues downstream leading us to propose the identity of the adrenal mitogen to be N-POMC (1-52).

Combinatorial peptide ligand libraries and plant proteomics: a winning strategy at a price

The mechanism of action and properties of a solid-phase ligand library made of hexapeptides (combinatorial peptide ligand libraries or CPLL, for capturing the "hidden proteome", i.e. the low- and very low-abundance proteins Constituting the vast majority of species in any proteome. as applied to plant tissues, are reviewed here. Plant tissues are notoriously recalcitrant to protein extraction and to proteome analysis, Firstly, rigid plant cell walls need to be mechanically disrupted to release the cell content and, in addition to their poor protein yield, plant tissues are rich in proteases and oxidative enzymes, contain phenolic Compounds, starches, oils, pigments and secondary metabolites that massively contaminate protein extracts. In addition, complex matrices of polysaccharides, including large amount of anionic pectins, are present. All these species compete with the binding of proteins to the CPLL beads, impeding proper capture and identification I detection of low-abundance species. When properly pre-treated, plant tissue extracts are amenable to capture by the CPLL beads revealing thus many new species among them low-abundance proteins. Examples are given on the treatment of leaf proteins, of corn seed extracts and of exudate proteins (latex from Hevea brasiliensis). In all cases, the detection of unique gene products via CPLL Capture is at least twice that of control, untreated sample. (c) 2008 Elsevier B.V. All rights reserved.

Proteome analysis for identification of target proteins of genistein in primary human endothelial cells stressed with oxidized LDL or homocysteine

Background Epidemiological studies suggest that soy consumption contributes to the prevention of coronary heart disease. The proposed anti-atherogenic effects of soy appear to be carried by the soy isoflavones with genistein as the most abundant compound. Aim of the study To identify proteins or pathways by which genistein might exert its protective activities on atherosclerosis, we analyzed the proteomic response of primary human umbilical vein endothelial cells ( HUVEC) that were exposed to the pro-atherosclerotic stressors homocysteine or oxidized low-density lipoprotein (ox-LDL). Methods HUVEC were incubated with physiological concentrations of homocysteine or ox-LDL in the absence and presence of genistein at concentrations that can be reached in human plasma by a diet rich in soy products (2.5 muM) or by pharmacological intervention ( 25 muM). Proteins from HUVEC were separated by two-dimensional polyacrylamide gel electrophoresis and those that showed altered expression level upon genistein treatment were identified by peptide mass fingerprints derived from tryptic digests of the protein spots. Results Several proteins were found to be differentially affected by genistein. The most interesting proteins that were potently decreased by homocysteine treatment were annexin V and lamin A. Annexin V is an antithrombotic molecule and mutations in nuclear lamin A have been found to result in perturbations of plasma lipids associated with hypertension. Genistein at low and high concentrations reversed the stressor-induced decrease of these anti-atherogenic proteins. Ox-LDL treatment of HUVEC resulted in an increase in ubiquitin conjugating enzyme 12, a protein involved in foam cell formation. Treatment with genistein at both doses reversed this effect. Conclusions Proteome analysis allows the identification of potential interactions of dietary components in the molecular process of atherosclerosis and consequently provides a powerful tool to define biomarkers of response.

Comparison of bottom-up protein identification methods

With the rapid development of proteomics, a number of different methods appeared for the basic task of protein identification. We made a simple comparison between a common liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow using an ion trap mass spectrometer and a combined LC-MS and LC-MS/MS method using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and accurate peptide masses. To compare the two methods for protein identification, we grew and extracted proteins from E. coli using established protocols. Cystines were reduced and alkylated, and proteins digested by trypsin. The resulting peptide mixtures were separated by reversed-phase liquid chromatography using a 4 h gradient from 0 to 50% acetonitrile over a C18 reversed-phase column. The LC separation was coupled on-line to either a Bruker Esquire HCT ion trap or a Bruker 7 tesla APEX-Qe Qh-FTICR hybrid mass spectrometer. Data-dependent Qh-FTICR-MS/MS spectra were acquired using the quadrupole mass filter and collisionally induced dissociation into the external hexapole trap. Proteins were in both schemes identified by Mascot MS/MS ion searches and the peptides identified from these proteins in the FTICR MS/MS data were used for automatic internal calibration of the FTICR-MS data, together with ambient polydimethylcyclosiloxane ions.

Modeling the tetraphenylalanine-PEG hybrid amphiphile: from DFT calculations on the peptide to molecular dynamics simulations on the conjugate

The conformational properties of the hybrid amphiphile formed by the conjugation of a hydrophobic peptide with four phenylalanine (Phe) residues and hydrophilic poly(ethylene glycol), have been investigated using quantum mechanical calculations and atomistic molecular dynamics simulations. The intrinsic conformational preferences of the peptide were examined using the building-up search procedure combined with B3LYP/ 6-31G(d) geometry optimizations, which led to the identification of 78, 78, and 92 minimum energy structures for the peptides containing one, two, and four Phe residues. These peptides tend to adopt regular organizations involving turn-like motifs that define ribbon or helicallike arrangements. Furthermore, calculations indicate that backbone ... side chain interactions involving the N-H of the amide groups and the pi clouds of the aromatic rings play a crucial role in Phe-containing peptides. On the other hand,MD simulations on the complete amphiphile in aqueous solution showed that the polymer fragment rapidly unfolds maximizing the contacts with the polar solvent, even though the hydrophobic peptide reduce the number of waters of hydration with respect to an individual polymer chain of equivalent molecular weight. In spite of the small effect of the peptide in the hydrodynamic properties of the polymer, we conclude that the two counterparts of the amphiphile tend to organize as independent modules.

Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease

A nitric oxide synthase (NOS)-like activity has been demonstrated in human red blood cells (RBCs), but doubts about its functional significance, isoform identity and disease relevance remain. Using flow cytometry in combination with the NO-imaging probe DAF-FM we find that all blood cells form NO intracellularly, with a rank order of monocytes > neutrophils > lymphocytes > RBCs > platelets. The observation of a NO-related fluorescence within RBCs was unexpected given the abundance of the NO-scavenger oxyhemoglobin. Constitutive normoxic NO formation was abolished by NOS inhibition and intracellular NO scavenging, confirmed by laser-scanning microscopy and unequivocally validated by detection of the DAF-FM reaction product with NO using HPLC and LC-MS/MS. Employing immunoprecipitation, ESI-MS/MS-based peptide sequencing and enzymatic assay we further demonstrate that human RBCs contain an endothelial NOS (eNOS) that converts L-3H-Arginine to L-3H-Citrulline in a Ca2+/Calmodulin-dependent fashion. Moreover, in patients with coronary artery disease, red cell eNOS expression and activity are both lower than in age-matched healthy individuals and correlate with the degree of endothelial dysfunction. Thus, human RBCs constitutively produce NO under normoxic conditions via an active eNOS isoform the activity of which is compromised in patients with coronary artery disease.