Formation of epsilon-formyllysine on silver-stained proteins: implications for assignment of isobaric dimethylation sites by tandem mass spectrometry.

Considerable effort is focused presently on the detection and comprehensive assignment of post-translational modifications of proteins. Obviously attention must be paid to the possibility of chemical modifications that may occur to protein samples during sample handling and manipulation prior to analysis by tandem mass spectrometry. This is of particular concern when a modification is isobaric with the mass differential in common with a known post-translational analog.

Isotope-coded and affinity-tagged cross-linking (ICATXL): an efficient strategy to probe protein interaction surfaces.

Chemical cross-linking followed by identification of the cross-linked residues by mass spectrometry provides structural information on protein interaction surfaces. Nevertheless, accurate analysis of the digested, cross-linked proteins is often challenging. Herein, we describe a novel strategy that relies on the use of affinity-tagged cross-linkers and isotope coding on the cross-linker-modified species.

Identification of novel quaternary domain interactions in the Hsp90 chaperone, GRP94.

The structural basis for the coupling of ATP binding and hydrolysis to chaperone activity remains a central question in Hsp90 biology. By analogy to MutL, ATP binding to Hsp90 is thought to promote intramolecular N-terminal dimerization, yielding a molecular clamp functioning in substrate protein activation. Though observed in studies with recombinant domains, whether such quaternary states are present in native Hsp90s is unknown.

Keratin 20 serine 13 phosphorylation is a stress and intestinal goblet cell marker.

Keratin polypeptide 20 (K20) is an intermediate filament protein with preferential expression in epithelia of the stomach, intestine, uterus, and bladder and in Merkel cells of the skin. K20 expression is used as a marker to distinguish metastatic tumor origin, but nothing is known regarding its regulation and function. We studied K20 phosphorylation as a first step toward understanding its physiologic role.

Warm ischemia-induced alterations in oxidative and inflammatory proteins in hepatic Kupffer cells in rats.

The aim of the study was to investigate the impact of ischemia/reperfusion injury on the proteome of Kupffer cells. Lean Zucker rats (n = 6 each group) were randomized to 75 min of warm ischemia or sham operation. After reperfusion for 8 h, Kupffer cells were isolated by enzymatic perfusion and density gradient centrifugation.

O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry.

O-GlcNAc is a widespread dynamic carbohydrate modification of cytosolic and nuclear proteins with features analogous to phosphorylation. O-GlcNAc acts critically in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression. However, the study of its specific regulatory functions has been limited by difficulties in mapping sites of O-GlcNAc modification.

Comprehensive identification of phosphorylation sites in postsynaptic density preparations.

In the mammalian central nervous system, the structure known as the postsynaptic density (PSD) is a dense complex of proteins whose function is to detect and respond to neurotransmitter released from presynaptic axon terminals. Regulation of protein phosphorylation in this molecular machinery is critical to the activity of its components, which include neurotransmitter receptors, kinases/phosphatases, scaffolding molecules, and proteins regulating cytoskeletal structure. To characterize the phosphorylation state of proteins in PSD samples, we combined strong cation exchange (SCX) chromatography with IMAC.

Peptidomics of a single identified neuron reveals diversity of multiple neuropeptides with convergent actions on cellular excitability.

In contrast to classical transmitters, the detailed structures and cellular and synaptic actions of neuropeptides are less well described. Peptide mass profiling of single identified neurons of the mollusc Lymnaea stagnalis indicated the presence of 17 abundant neuropeptides in the cardiorespiratory neuron, visceral dorsal 1 (VD1), and a subset of 14 peptides in its electrically coupled counterpart, right parietal dorsal 2. Altogether, based on this and previous work, we showed that the high number of peptides arises from the expression and processing of four distinct peptide precursor proteins, including a novel one.

Mapping post-translational modifications of the histone variant MacroH2A1 using tandem mass spectrometry.

Post-translational histone modifications modulate chromatin-templated processes and therefore affect cellular proliferation, growth, and development. Although post-translational modifications on the core histones have been under intense investigation for several years, the modifications on variant histones are poorly understood. We used tandem mass spectrometry to identify covalent modifications on a histone H2A variant, macroH2A1.

Neurotransmitter release regulated by a MALS-liprin-alpha presynaptic complex.

Synapses are highly specialized intercellular junctions organized by adhesive and scaffolding molecules that align presynaptic vesicular release with postsynaptic neurotransmitter receptors. The MALS/Veli-CASK-Mint-1 complex of PDZ proteins occurs on both sides of the synapse and has the potential to link transsynaptic adhesion molecules to the cytoskeleton. In this study, we purified the MALS protein complex from brain and found liprin-alpha as a major component.

Atg19p ubiquitination and the cytoplasm to vacuole trafficking pathway in yeast.

The cytoplasm to vacuole (Cvt) trafficking pathway in S. cerevisiae is a constitutive biosynthetic pathway required for the transport of two vacuolar enzymes, aminopeptidase I (Ape1p) and alpha-mannosidase (Ams1p), to the vacuole. Ape1p and Ams1p bind to their receptor, Atg19p, in the cytosol to form a Cvt complex, which then associates with a membrane structure that envelops the complex before fusing with the vacuolar membrane.

Analysis of leaf proteome after UV-B irradiation in maize lines differing in sensitivity.

UV-B radiation causes diverse morphological and physiological responses in plants, but the underlying mechanisms governing these integrated responses are unknown. In this study, we systematically surveyed responses of maize leaves to UV-B radiation using DIGE 2D gels and identified selected proteins by mass spectrometry and immunodetection analysis. To identify changes in protein accumulation in response to UV-B radiation, a line (b, pl W23) deficient in flavonoid sunscreen compounds and hence similar to commercial corn was used.

From proteins to proteomics.

During the second half of the 20th century, biochemistry and subsequently molecular biology blossomed into the core upon which all biological and biomedical sciences now depend. A major part of these closely related disciplines has been the study of the structure and function of proteins and the diverse biological functions that they perform. Early experimentation necessarily focused on individual entities, selected mainly for their activities, but as technology improved there developed a tendency to look at proteins as larger, interactive groups or clusters.

WDR5 associates with histone H3 methylated at K4 and is essential for H3 K4 methylation and vertebrate development.

Histone H3 lysine 4 (K4) methylation has been linked to the transcriptional activation in a variety of eukaryotic species. Here we show that a common component of MLL1, MLL2, and hSet1 H3 K4 methyltransferase complexes, the WD40-repeat protein WDR5, directly associates with histone H3 di- and trimethylated at K4 and with H3-K4-dimethylated nucleosomes. WDR5 is required for binding of the methyltransferase complex to the K4-dimethylated H3 tail as well as for global H3 K4 trimethylation and HOX gene activation in human cells.

Comprehensive analysis of a multidimensional liquid chromatography mass spectrometry dataset acquired on a quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometer: II. New developments in Protein Prospector allow for reliable and comprehensive automatic analysis of large datasets.

A thorough analysis of the protein interaction partners of the yeast GTPase Gsp1p was carried out by a multidimensional chromatography strategy of strong cation exchange fractionation of peptides followed by reverse phase LC-ESI-MSMS using a QSTAR instrument. This dataset was then analyzed using the latest developmental version of Protein Prospector. The Prospector search results were also compared with results from the search engine "Mascot" using a new results comparison program within Prospector named "SearchCompare.

Comprehensive analysis of a multidimensional liquid chromatography mass spectrometry dataset acquired on a quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometer: I. How much of the data is theoretically interpretable by search engines?

An in-depth analysis of a multidimensional chromatography-mass spectrometry dataset acquired on a quadrupole selecting, quadrupole collision cell, time-of-flight (QqTOF) geometry instrument was carried out. A total of 3269 CID spectra were acquired. Through manual verification of database search results and de novo interpretation of spectra 2368 spectra could be confidently determined as predicted tryptic peptides.

Molecular constituents of neuronal AMPA receptors.

Dynamic regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) underlies aspects of synaptic plasticity. Although numerous AMPAR-interacting proteins have been identified, their quantitative and relative contributions to native AMPAR complexes remain unclear. Here, we quantitated protein interactions with neuronal AMPARs by immunoprecipitation from brain extracts.

An unbiased cell morphology-based screen for new, biologically active small molecules.

We have implemented an unbiased cell morphology-based screen to identify small-molecule modulators of cellular processes using the Cytometrix (TM) automated imaging and analysis system. This assay format provides unbiased analysis of morphological effects induced by small molecules by capturing phenotypic readouts of most known classes of pharmacological agents and has the potential to read out pathways for which little is known. Four human-cancer cell lines and one noncancerous primary cell type were treated with 107 small molecules comprising four different protein kinase-inhibitor scaffolds.

Quantitative analysis of both protein expression and serine / threonine post-translational modifications through stable isotope labeling with dithiothreitol.

While phosphorylation and O-GlcNAc (cytoplasmic and nuclear glycosylation) are linked to normal and pathological changes in cell states, these post-translational modifications have been difficult to analyze in proteomic studies. We describe advances in beta-elimination / Michael addition-based approaches which allow for mass spectrometry-based identification and comparative quantification of O-phosphate or O-GlcNAc-modified peptides, as well as cysteine-containing peptides for expression analysis. The method (BEMAD) involves differential isotopic labeling through Michael addition with normal dithiothreitol (DTT) (d0) or deuterated DTT (d6), and enrichment of these peptides by thiol chromatography.

Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex.

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied.

Proteome changes in ovarian epithelial cells derived from women with BRCA1 mutations and family histories of cancer.

Malignant transformation of the ovarian surface epithelium (OSE) accounts for most ovarian carcinoma. Detection of preneoplastic changes in the OSE leading to overt malignancy is important in prevention and management of ovarian cancer. We identified OSE proteins with altered expression derived from women with a family history (FH) of ovarian and/or breast cancer and mutations in the BRCA1 tumor suppressor gene.

Unraveling the interface of signal recognition particle and its receptor by using chemical cross-linking and tandem mass spectrometry.

Among the methods used to unravel protein interaction surfaces, chemical cross-linking followed by identification of the cross-linked peptides by mass spectrometry has proven especially useful in dynamic and complex systems. During the signal recognition particle (SRP)-dependent targeting of proteins to the bacterial plasma membrane, the specific interaction between Ffh (the protein component of SRP) and FtsY (the SRP receptor) is known to be essential for the efficiency and fidelity of this process. In this work, we studied the Escherichia coli and Thermus aquaticus Ffh.

The mammalian retromer regulates transcytosis of the polymeric immunoglobulin receptor.

Epithelial cells have separate apical and basolateral plasma membrane domains with distinct compositions. After delivery to one surface, proteins can be endocytosed and then recycled, degraded or transcytosed to the opposite surface. Proper sorting into the transcytotic pathway is essential for maintaining polarity, as most proteins are endocytosed many times during their lifespan.