Differential proteomic comparison of breast cancer secretome using a quantitative paired analysis workflow.

Background: Worldwide, breast cancer is the main cause of cancer mortality in women. Most cases originate in mammary ductal cells that produce the nipple aspirate fluid (NAF). In cancer patients, this secretome contains proteins associated with the tumor microenvironment.

Biochemical and pathological changes result from mutated Caveolin-3 in muscle.

Background: Caveolin-3 (CAV3) is a muscle-specific protein localized to the sarcolemma. It was suggested that CAV3 is involved in the connection between the extracellular matrix (ECM) and the cytoskeleton. Caveolinopathies often go along with increased CK levels indicative of sarcolemmal damage.

The mTOR and PP2A Pathways Regulate PHD2 Phosphorylation to Fine-Tune HIF1α Levels and Colorectal Cancer Cell Survival under Hypoxia.

Oxygen-dependent HIF1α hydroxylation and degradation are strictly controlled by PHD2. In hypoxia, HIF1α partly escapes degradation because of low oxygen availability. Here, we show that PHD2 is phosphorylated on serine 125 (S125) by the mechanistic target of rapamycin (mTOR) downstream kinase P70S6K and that this phosphorylation increases its ability to degrade HIF1α.

Simultaneous Metabolite, Protein, Lipid Extraction (SIMPLEX): A Combinatorial Multimolecular Omics Approach for Systems Biology.

Interconnected molecular networks are at the heart of signaling pathways that mediate adaptive plasticity of eukaryotic cells. To gain deeper insights into the underlying molecular mechanisms, a comprehensive and representative analysis demands a deep and parallel coverage of a broad spectrum of molecular species. Therefore, we introduce a simultaneous metabolite, protein, lipid extraction (SIMPLEX) procedure, a novel strategy for the quantitative investigation of lipids, metabolites, and proteins.

Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro.

Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation.

Multidimensional electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) for quantitative analysis of the proteome and phosphoproteome in clinical and biomedical research.

Quantitative proteomics and phosphoproteomics have become key disciplines in understanding cellular processes. Fundamental research can be done using cell culture providing researchers with virtually infinite sample amounts. In contrast, clinical, pre-clinical and biomedical research is often restricted to minute sample amounts and requires an efficient analysis with only micrograms of protein.

Highly sensitive phosphoproteomics by tailoring solid-phase extraction to electrostatic repulsion-hydrophilic interaction chromatography.

In the past decade, several strategies for comprehensive phosphoproteome analysis have been introduced. Most of them combine different phosphopeptide enrichment techniques and require starting material in the milligram range, as a consequence of their insufficient sensitivity. This limitation impairs the applicability of phosphoproteomics to a wide variety of clinical research, where sample material is highly limited.

Myopathy in Marinesco-Sjögren syndrome links endoplasmic reticulum chaperone dysfunction to nuclear envelope pathology.

Marinesco-Sjögren syndrome (MSS) features cerebellar ataxia, mental retardation, cataracts, and progressive vacuolar myopathy with peculiar myonuclear alterations. Most MSS patients carry homozygous or compound heterozygous SIL1 mutations. SIL1 is a nucleotide exchange factor for the endoplasmic reticulum resident chaperone BiP which controls a plethora of essential processes in the endoplasmic reticulum.

Comprehensive mutational analysis reveals p6Gag phosphorylation to be dispensable for HIV-1 morphogenesis and replication.

The structural polyprotein Gag of human immunodeficiency virus type 1 (HIV-1) is necessary and sufficient for formation of virus-like particles. Its C-terminal p6 domain harbors short peptide motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. p6 has been shown to be the major viral phosphoprotein in HIV-1-infected cells and virions, but the sites and functional relevance of p6 phosphorylation are not clear.

Revealing phosphoproteins playing role in tobacco pollen activated in vitro.

The transition between the quiescent mature and the metabolically active germinating pollen grain most probably involves changes in protein phosphorylation status, since phosphorylation has been implicated in the regulation of many cellular processes. Given that, only a minor proportion of cellular proteins are phosphorylated at any one time, and that phosphorylated and nonphosphorylated forms of many proteins can co-exist within a cell, the identification of phosphoproteins requires some prior enrichment from a crude protein extract. Here, we have used metal oxide/hydroxide affinity chromatography (MOAC) based on an aluminum hydroxide matrix for this purpose, and have generated a population of phosphoprotein candidates from both mature and in vitro activated tobacco pollen grains.

iTRAQ data interpretation.

Quantitative proteomic analysis can help elucidating unexplored biological questions; it, however, relies on highly reproducible experiments and reliable data processing. Among the existing strategies, iTRAQ is known as an easy to use method allowing relative comparison of up to eight multiplexed samples.Once the data is acquired it is important that the final protein quantification reflects the actual amounts in the samples.

Robust workflow for iTRAQ-based peptide and protein quantification.

Quantitative proteomics has become a routinely used technique to globally compare protein content and expression profiles of biological samples, for instance after differential stimulation. In this context, chemical stable isotope-based labeling techniques, such as ICAT and iTRAQ, have been successfully applied in a large variety of studies. Since iTRAQ labels are isobaric, quantitation is conducted on the MS/MS level.

Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MS-based proteomics.

Trypsin is the most frequently used proteolytic enzyme in mass spectrometry-based proteomics. Beside its good availability, it also offers some major advantages such as an optimal average peptide length of ~14 amino acids, and typically the presence of at least two defined positive charges at the N-terminus as well as the C-terminal Arg/Lys, rendering tryptic peptides well suited for CID-based LC-MS/MS. Here, we conducted a systematic study of different types of commercially available trypsin in order to qualitatively and quantitatively compare cleavage specificity, efficiency as well as reproducibility and the potential impact on quantitation and proteome coverage.

Phosphoproteome analysis of the platelet plasma membrane.

Blood platelets are key players standing at the crossroads between physiologically occurring hemostasis and pathologic thrombus formation. As these cellular particles lack a nucleus, intra- and intercellular processes involved in platelet activity and function are almost exclusively regulated on the protein level. In particular, posttranslational protein modification by phosphorylation, which allows for a quick and highly dynamic transduction of cellular signals, is discussed in this context.

The good, the bad, the ugly: validating the mass spectrometric analysis of modified peptides.

Mass spectrometric characterization of protein modifications is usually based on single peptides. With the advent of large-scale PTM-focussed MS studies, vast amounts of data are generated continuously, providing biologists extremely valuable and virtually never-ending sources for targeted functional research. However, even more than for proteomics in general, appropriate strategies for quality control of the different steps of the analytical strategy are imperative to prevent functional researchers from doing Sisyphos work on false-positive and unconfident PTM assignments.

Catch me if you can: mass spectrometry-based phosphoproteomics and quantification strategies.

Phosphorylation of proteins is one of the most prominent PTMs and for instance a key regulator of signal transduction. In order to improve our understanding of cellular phosphorylation events, considerable effort has been devoted to improving the analysis of phosphorylation by MS-based proteomics. Different enrichment strategies for phosphorylated peptides/proteins, such as immunoaffinity chromatography (IMAC) or titanium dioxide, have been established and constantly optimized for subsequent MS analysis.

Recent advances in yeast organelle and membrane proteomics.

Yeast proteome research comprises two different aspects: with respect to systemic fungal infections (fungemias), invasive candidiasis, for instance by Candida albicans, is among the most common causes of morbidity and mortality particularly in the expanding population of immunocompromised patients, which rises a high medical and pharmaceutical interest in this facultative pathogenic organism. Apart from its clinical relevance, yeast research moreover provides an indispensable source of knowledge regarding fundamental biochemical processes of eukaryotic cells. In this context, the budding yeast Saccharomyces cerevisiae is, in addition to its multiple industrial applications, one of the most extensively used microorganisms and serves as the best understood eukaryotic model system so far.

Assembly of nuclear pore complexes mediated by major vault protein.

During interphase growth of eukaryotic cells, nuclear pore complexes (NPCs) are continuously incorporated into the intact nuclear envelope (NE) by mechanisms that are largely unknown. De novo formation of NPCs involves local fusion events between the inner and outer nuclear membrane, formation of a transcisternal membranous channel of defined diameter and the coordinated assembly of hundreds of nucleoporins into the characteristic NPC structure. Here we have used a cell-free system based on Xenopus egg extract, which allows the experimental separation of nuclear-membrane assembly and NPC formation.

The human platelet membrane proteome reveals several new potential membrane proteins.

We present the first focused proteome study on human platelet membranes. Due to the removal of highly abundant cytoskeletal proteins a wide spectrum of known platelet membrane proteins and several new and hypothetical proteins were accessible. In contrast to other proteome studies we focused on prefractionation and purification of membranes from human platelets according to published protocols to reduce sample complexity and enrich interesting membrane proteins.

Two-dimensional benzyldimethyl-n-hexadecylammonium chloride/SDS-PAGE for membrane proteomics.

Despite the importance of membranes in any living system, the global analysis of membrane subproteomes is still a common obstacle. In particular, the widely used 2-DE technique consisting of IEF in the first dimension and SDS-PAGE in the second dimension has some major drawbacks regarding the separation of hydrophobic proteins. Therefore, we applied an alternative electrophoretic technique for separating membrane proteins: two-dimensional BAC/SDS electrophoresis (2-DB) using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride in the first and the anionic detergent SDS in the second dimension.