Proteome analysis and conditional deletion of the EAAT2 glutamate transporter provide evidence against a role of EAAT2 in pancreatic insulin secretion in mice.

Islet function is incompletely understood in part because key steps in glutamate handling remain undetermined. The glutamate (excitatory amino acid) transporter 2 (EAAT2; Slc1a2) has been hypothesized to (a) provide islet cells with glutamate, (b) protect islet cells against high extracellular glutamate concentrations, (c) mediate glutamate release, or (d) control the pH inside insulin secretory granules. Here we floxed the EAAT2 gene to produce the first conditional EAAT2 knock-out mice. Crossing with Nestin-cyclization recombinase (Cre) eliminated EAAT2 from the brain, resulting in epilepsy and premature death, confirming the importance of EAAT2 for brain function and validating the genetic construction. Crossing with insulin-Cre lines (RIP-Cre and IPF1-Cre) to obtain pancreas-selective deletion did not appear to affect survival, growth, glucose tolerance, or ß-cell number. We found (using TaqMan RT-PCR, immunoblotting, immunocytochemistry, and proteome analysis) that the EAAT2 levels were too low to support any of the four hypothesized functions. The proteome analysis detected more than 7,000 islet proteins of which more than 100 were transporters. Although mitochondrial glutamate transporters and transporters for neutral amino acids were present at high levels, all other transporters with known ability to transport glutamate were strikingly absent. Glutamate-metabolizing enzymes were abundant. The level of glutamine synthetase was 2 orders of magnitude higher than that of glutaminase. Taken together this suggests that the uptake of glutamate by islets from the extracellular fluid is insignificant and that glutamate is intracellularly produced. Glutamine synthetase may be more important for islets than assumed previously.

Quantitative proteomic analysis of single pancreatic islets.

Technological developments make mass spectrometry (MS)-based proteomics a central pillar of biochemical research. MS has been very successful in cell culture systems, where sample amounts are not limiting. To extend its capabilities to extremely small, physiologically distinct cell types isolated from tissue, we developed a high sensitivity chromatographic system that measures nanogram protein mixtures for 8 h with very high resolution. This technology is based on splitting gradient effluents into a capture capillary and provides an inherent technical replicate. In a single analysis, this allowed us to characterize kidney glomeruli isolated by laser capture microdissection to a depth of more than 2,400 proteins. From pooled pancreatic islets of Langerhans, another type of "miniorgan," we obtained an in-depth proteome of 6,873 proteins, many of them involved in diabetes. We quantitatively compared the proteome of single islets, containing 2,000-4,000 cells, treated with high or low glucose levels, and covered most of the characteristic functions of beta cells. Our ultrasensitive analysis recapitulated known hyperglycemic changes but we also find components up-regulated such as the mitochondrial stress regulator Park7. Direct proteomic analysis of functionally distinct cellular structures opens up perspectives in physiology and pathology.

A novel chromatographic method allows on-line reanalysis of the proteome.

Liquid chromatography combined with electrospray ionization is widely used for direct analysis of polar and labile molecules by LCMS. The on-line coupling in LCMS is a major strength but also causes a principal limitation that each eluting analyte has to be analyzed immediately and is not available for detailed interrogation after the LCMS run. Here we developed a new chromatographic strategy, which removes this limitation. After column separation the flow is split, one portion is analyzed directly, and the other is diverted to a capture capillary. After the direct LCMS run, the flow is switched, and the portion stored in the capillary is analyzed ("replay run"). We describe a setup consisting of an analytical column, a splitting valve, and a focusing column, which performs at full sensitivity and undiminished chromatographic resolution. We demonstrate three principal advantages of this system: nearly continuous MS utilization, duplicate analysis without requirement for additional sample, and targeting of important but undersampled features in the replay run.

Top-down quantitation and characterization of SILAC-labeled proteins.

Stable isotope labeling by amino acids in cell culture (SILAC) has become a popular labeling strategy for peptide quantitation in proteomics experiments. If the SILAC technology could be extended to intact proteins, it would enable direct quantitation of their relative expression levels and of the degree of modification between different samples. Here we show through modeling and experiments that SILAC is suitable for intact protein quantitation and top-down characterization. When SILAC-labeling lysine and/or arginine, peaks of light and heavy SILAC-doublets do not interfere with peaks of different charge states at least between 10 and 200 kDa. Unlike chemical methods, SILAC ensures complete incorporation-all amino acids are labeled. The isotopic enrichment of commercially available SILAC amino acids of nominally 95% to 98% shifts the mass difference between light and heavy state but does not lead to appreciably broadened peaks. We expressed labeled and unlabeled Grb2, a 28 kDa signaling protein, and showed that the two forms can be quantified with an average standard deviation of 6%. We performed on-line top-down sequencing of both forms in a hybrid linear ion trap orbitrap instrument. The quantized mass offset between fragments provided information about the number of labeled residues in the fragments, thereby simplifying protein identification and characterization.

Circulating biomarkers for predicting cardiovascular disease risk; a systematic review and comprehensive overview of meta-analyses.

BACKGROUND: Cardiovascular disease is one of the major causes of death worldwide. Assessing the risk for cardiovascular disease is an important aspect in clinical decision making and setting a therapeutic strategy, and the use of serological biomarkers may improve this. Despite an overwhelming number of studies and meta-analyses on biomarkers and cardiovascular disease, there are no comprehensive studies comparing the relevance of each biomarker. We performed a systematic review of meta-analyses on levels of serological biomarkers for atherothrombosis to compare the relevance of the most commonly studied biomarkers. METHODS AND FINDINGS: Medline and Embase were screened on search terms that were related to "arterial ischemic events" and "meta-analyses". The meta-analyses were sorted by patient groups without pre-existing cardiovascular disease, with cardiovascular disease and heterogeneous groups concerning general populations, groups with and without cardiovascular disease, or miscellaneous. These were subsequently sorted by end-point for cardiovascular disease or stroke and summarized in tables. We have identified 85 relevant full text articles, with 214 meta-analyses. Markers for primary cardiovascular events include, from high to low result: C-reactive protein, fibrinogen, cholesterol, apolipoprotein B, the apolipoprotein A/apolipoprotein B ratio, high density lipoprotein, and vitamin D. Markers for secondary cardiovascular events include, from high to low result: cardiac troponins I and T, C-reactive protein, serum creatinine, and cystatin C. For primary stroke, fibrinogen and serum uric acid are strong risk markers. Limitations reside in that there is no acknowledged search strategy for prognostic studies or meta-analyses. CONCLUSIONS: For primary cardiovascular events, markers with strong predictive potential are mainly associated with lipids. For secondary cardiovascular events, markers are more associated with ischemia. Fibrinogen is a strong predictor for primary stroke.

Top-down protein sequencing and MS3 on a hybrid linear quadrupole ion trap-orbitrap mass spectrometer.

Top-down proteomics, the analysis of intact proteins (instead of first digesting them to peptides), has the potential to become a powerful tool for mass spectrometric protein characterization. Requirements for extremely high mass resolution, accuracy, and ability to efficiently fragment large ions have often limited top-down analyses to custom built FT-ICR mass analyzers. Here we explore the hybrid linear ion trap (LTQ)-Orbitrap, a novel, high performance, and compact mass spectrometric analyzer, for top-down proteomics. Protein standards from 10 to 25 kDa were electrosprayed into the instrument using a nanoelectrospray chip. Resolving power of 60,000 was ample for isotope resolution of all protein charge states. We achieved absolute mass accuracies for intact proteins between 0.92 and 2.8 ppm using the "lock mass" mode of operation. Fifty femtomole of cytochrome c applied to the chip resulted in spectra with excellent signal-to-noise ratio and only low attomole sample consumption. Different protein charge states were dissociated in the LTQ, and the sensitivity of the orbitrap allowed routine, high resolution, and high mass accuracy fragment detection. This resulted in unambiguous charge state determination of fragment ions and identification of unmodified and modified proteins by database searching. Protein fragments were further isolated and fragmented in the LTQ followed by analysis of MS(3) fragments in the orbitrap, localizing modifications to part of the sequence and helping to identify the protein with these small peptide-like fragments. Given the ready availability and ease of operation of the LTQ-Orbitrap, it may have significant impact on top-down proteomics.