Jens R. Coorssen

De Novo Sequencing of Peptides Using MALDI/TOF-TOF

The recently developed MALDI TOF-TOF instrument yields relatively complex but interpretable fragmentation spectra. When coupled with a straightforward sequence extension algorithm, it is possible to develop complete peptide sequences de novo from the spectra. This approach has been applied to a set of peptides derived from typtic digestion of electrophoretically separated sea urchin egg membrane proteins. When directed to proteins that have been described previously, the results were in essential agreement with those obtained by conventional data base searching approaches, with certain important exceptions. The present method detected errors in published sequences and was able to develop sequences from peptides differing in mass by one dalton (Da). These results show both the power of the present approach and the need for using de novo methods more frequently than may be otherwise appreciated.

Cholesterol facilitates the native mechanism of Ca2+-triggered membrane fusion

The process of regulated exocytosis is defined by the Ca2+-triggered fusion of two apposed membranes, enabling the release of vesicular contents. This fusion step involves a number of energetically complex steps and requires both protein and lipid membrane components. The role of cholesterol has been investigated using isolated release-ready native cortical secretory vesicles to analyze the Ca2+-triggered fusion step of exocytosis. Cholesterol is a major component of vesicle membranes and we show here that selective removal from membranes, selective sequestering within membranes, or enzymatic modification causes a significant inhibition of the extent, Ca2+ sensitivity and kinetics of fusion. Depending upon the amount incorporated, addition of exogenous cholesterol to cholesterol-depleted membranes consistently recovers the extent, but not the Ca2+ sensitivity or kinetics of fusion. Membrane components of comparable negative curvature selectively recover the ability to fuse, but are unable to recover the kinetics and Ca2+ sensitivity of vesicle fusion. This indicates at least two specific positive roles for cholesterol in the process of membrane fusion: as a local membrane organizer contributing to the efficiency of fusion, and, by virtue of its intrinsic negative curvature, as a specific molecule working in concert with protein factors to facilitate the minimal molecular machinery for fast Ca2+-triggered fusion.

Ca2+ triggers massive exocytosis in Chinese hamster ovary cells.

We have tracked the cell surface area of CHO cells by measuring the membrane capacitance, Cm. An increase in cytosolic [Ca2+], [Ca2+]i, increased the cell surface area by 20–30%. At micromolar [Ca2+]i the increase occurred in minutes, while at 20 microM or higher [Ca2+]i it occurred in seconds and was transient. GTPgammaS caused a 3% increase even at 0.1 microM [Ca2+]i. We conclude that CHO cells, previously thought capable only of constitutive exocytosis, can perform Ca2+‐triggered exocytosis that is both massive and rapid. Ca2+‐triggered exocytosis was also observed in 3T3 fibroblasts. Our findings add evidence to the view that Ca induces exocytosis in cells other than known secretory cells.

Fusion pore expansion in horse eosinophils is modulated by Ca2+ and protein kinase C via distinct mechanisms.

Using the patch‐clamp technique, we studied the role of protein phosphorylation and dephosphorylation on the exocytotic fusion of secretory granules with the plasma membrane in horse eosinophils. Phorbol 12‐myristate 13‐acetate (PMA) had no effect on the amplitude and dynamics of degranulation, indicating that the formation of fusion pores is insensitive to activation of protein kinase C (PKC). Fusion pore expansion, however, was accelerated ∼2‐fold by PMA, and this effect was abolished by staurosporine. Elevating intracellular Ca2+ to 1.5 μM also resulted in a 2‐fold acceleration of pore expansion; this effect was not prevented by staurosporine, indicating that intracellular Ca2+ and activation of PKC accelerate fusion pore expansion via distinct mechanisms. However, fusion pores can expand fully even when PKC is inhibited. In contrast, the phosphatase inhibitor α‐naphthylphosphate inhibits exocytotic fusion and slows fusion pore expansion. These results demonstrate that, subsequent to its formation, fusion pore expansion is under control of proteins subject to functional changes based on their phosphorylation states.

2DE : the phoenix of proteomics

UNLABELLED Given the rapid developments in mass spectrometry (MS) in terms of sensitivity, mass accuracy, and throughput, some have suggested that two-dimensional gel electrophoresis (2DE) may no longer be a method of choice for proteomic analyses. However, as recognition of issues with these newer shotgun-MS approaches grows, there is a fresh and growing regard for the maturity of 2DE-MS as a genuine top-down analytical approach, particularly as it resolves thousands of intact protein species in a single run, enabling the simultaneous analysis of total protein complement, including isoforms and post-translational modifications. Given the strengths of both, it is most appropriate to view these as complementary or at least parallel approaches: as proteins encompass a myriad of physico-chemical properties, and the real aim is to explore proteomes as deeply as possible, all available resolving strategies must be considered in terms of the complexity encountered. It is time to critically and constructively focus on the optimization and integration of existing techniques rather than simplistically suggesting that one should replace the other. Our intention here is thus to present an overview of protein resolving techniques, focusing on milestones associated with 2DE, including pros, cons, advances and variations, in particular relative to shotgun proteomic approaches. BIOLOGICAL SIGNIFICANCE Proteomic researchers recognize the importance of 2DE in the history of proteomics. But the latest developments in mass spectrometry-based techniques have led some researchers to retire 2DE in their labs. However, we argue here that 2DE-MS is a genuine top-down analytical approach. The significance of this discussion is to make proteomic researchers aware of the importance of this technique in a proteomic pipeline. This article is part of a Special Issue entitled: Environmental and structural proteomics.

Quantitative proteomics : assessing the spectrum of in-gel protein detection methods

Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.

Enhanced detergent extraction for analysis of membrane proteomes by two-dimensional gel electrophoresis

BackgroundThe analysis of hydrophobic membrane proteins by two-dimensional gel electrophoresis has long been hampered by the concept of inherent difficulty due to solubility issues. We have optimized extraction protocols by varying the detergent composition of the solubilization buffer with a variety of commercially available non-ionic and zwitterionic detergents and detergent-like phospholipids.ResultsAfter initial analyses by one-dimensional SDS-PAGE, quantitative two-dimensional analyses of human erythrocyte membranes, mouse liver membranes, and mouse brain membranes, extracted with buffers that included the zwitterionic detergent MEGA 10 (decanoyl-N-methylglucamide) and the zwitterionic lipid LPC (1-lauroyl lysophosphatidylcholine), showed selective improvement over extraction with the common 2-DE detergent CHAPS (3 [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Mixtures of the three detergents showed additive improvements in spot number, density, and resolution. Substantial improvements in the analysis of a brain membrane proteome were observed.ConclusionThis study demonstrates that an optimized detergent mix, coupled with rigorous sample handling and electrophoretic protocols, enables simple and effective analysis of membrane proteomes using two-dimensional electrophoresis.

Regulated secretion: SNARE density, vesicle fusion and calcium dependence

SNAREs such as VAMP, SNAP-25 and syntaxin are essential for intracellular trafficking, but what are their exact molecular roles and how are their interactions with other proteins manifest? Capitalizing on the differential sensitivity of SNAREs to exogenous proteases, we quantified the selective removal of identified SNAREs from native secretory vesicles without loss of fusion competence. Using previously established fusion assays and a high sensitivity immunoblotting protocol, we analyzed the relationship between these SNARE proteins and Ca2+-triggered membrane fusion. Neither the extent of fusion nor the number of intermembrane fusion complexes per vesicle were correlated with the measured density of identified egg cortical vesicle (CV) SNAREs. Without syntaxin, CVs remained fusion competent. Surprisingly, for one (but not another) protease the Ca2+ dependence of fusion was correlated with CV SNARE density, suggesting a native protein complex that associates with SNAREs, the architecture of which ensures high Ca2+ sensitivity. As SNAREs may function during CV docking in vivo, and as further proteolysis after SNARE removal eventually ablates fusion, we hypothesize that the triggered steps of regulated fusion (Ca2+ sensitivity and the catalysis and execution of fusion) require additional proteins that function downstream of SNAREs.

Cholesterol, regulated exocytosis and the physiological fusion machine

Exocytosis is a highly conserved and essential process. Although numerous proteins are involved throughout the exocytotic process, the defining membrane fusion step appears to occur through a lipid-dominated mechanism. Here we review and integrate the current literature on protein and lipid roles in exocytosis, with emphasis on the multiple roles of cholesterol in exocytosis and membrane fusion, in an effort to promote a more molecular systems-level view of the as yet poorly understood process of Ca2+-triggered membrane mergers.

Transcriptome analysis of the central nervous system of the mollusc Lymnaea stagnalis

BackgroundThe freshwater snail Lymnaea stagnalis (L. stagnalis) has served as a successful model for studies in the field of Neuroscience. However, a serious drawback in the molecular analysis of the nervous system of L. stagnalis has been the lack of large-scale genomic or neuronal transcriptome information, thereby limiting the use of this unique model.ResultsIn this study, we report 7,712 distinct EST sequences (median length: 847 nucleotides) of a normalized L. stagnalis central nervous system (CNS) cDNA library, resulting in the largest collection of L. stagnalis neuronal transcriptome data currently available. Approximately 42% of the cDNAs can be translated into more than 100 consecutive amino acids, indicating the high quality of the library. The annotated sequences contribute 12% of the predicted transcriptome size of 20,000. Surprisingly, approximately 37% of the L. stagnalis sequences only have a tBLASTx hit in the EST library of another snail species Aplysia californica (A. californica) even using a low stringency e-value cutoff at 0.01. Using the same cutoff, approximately 67% of the cDNAs have a BLAST hit in the NCBI non-redundant protein and nucleotide sequence databases (nr and nt), suggesting that one third of the sequences may be unique to L. stagnalis. Finally, using the same cutoff (0.01), more than half of the cDNA sequences (54%) do not have a hit in nematode, fruitfly or human genome data, suggesting that the L. stagnalis transcriptome is significantly different from these species as well. The cDNA sequences are enriched in the following gene ontology functional categories: protein binding, hydrolase, transferase, and catalytic enzymes.ConclusionThis study provides novel molecular insights into the transcriptome of an important molluscan model organism. Our findings will contribute to functional analyses in neurobiology, and comparative evolutionary biology. The L. stagnalis CNS EST database is available at http://www.Lymnaea.org/.