Evaluation of reproducibility of protein identification results after multidimensional human serum protein separation

We developed a gel- and label-free proteomics platform for comparative studies of human serum. The method involves the depletion of the six most abundant proteins, protein fractionation by Off-Gel IEF and RP-HPLC, followed by tryptic digestion, LC-MS/MS, protein identification, and relative quantification using probabilistic peptide match score summation (PMSS). We evaluated performance and reproducibility of the complete platform and the individual dimensions, by using chromatograms of the RP-HPLC runs, PMSS based abundance scores and abundance distributions as objective endpoints. We were interested if a relationship exists between the quantity ratio and the PMSS score ratio. The complete analysis was performed four times with two sets of serum samples containing different concentrations of spiked bovine beta-lactoglobulin (0.1 and 0.3%, w/w). The two concentrations resulted in significantly differing PMSS scores when compared to the variability in PMSS scores of all other protein identifications. We identified 196 proteins, of which 116 were identified four times in corresponding fractions whereof 73 qualified for relative quantification. Finally, we characterized the PMSS based protein abundance distributions with respect to the two dimensions of fractionation and discussed some interesting patterns representing discrete isoforms. We conclude that combination of Off-Gel electrophoresis (OGE) and HPLC is a reproducible protein fractionation technique, that PMSS is applicable for relative quantification, that the number of quantifiable proteins is always smaller than the number of identified proteins and that reproducibility of protein identifications should supplement probabilistic acceptance criteria.

Microgram-Level Radiocarbon Determination of Carbonaceous Particles in Firn and Ice Samples: Pretreatment and Oc/Ec Separation

Carbonaceous particles that comprise organic carbon (OC) and elemental carbon (EC) are of increasing interest in climate research because of their influence on the radiation balance of the Earth. The radiocarbon determination of particulate OC and EC extracted from ice cores provides a powerful tool to reconstruct the long-term natural and anthropogenic emissions of carbonaceous particles. However, this C-14-based source apportionment method has not been applied for the firn section, which is the uppermost part of Alpine glaciers with a typical thickness of up to 50 m. In contrast to glacier ice, firn samples are more easily contaminated through drilling and handling operations. In this study, an alternative decontamination method for firn samples consisting of chiselling off the outer parts instead of rinsing them was developed and verified. The obtained procedural blank of 2.8 +/- 0.8 mu g C for OC is a factor of 2 higher compared to the rinsing method used for ice, but still relatively low compared to the typical OC concentration in firn samples from Alpine glaciers. The EC blank of 0.3 +/- 0.1 mu g C is similar for both methods. For separation of OC and EC for subsequent C-14 analysis, a thermal-optical method instead of the purely thermal method was applied for the first time to firn and ice samples, resulting in a reduced uncertainty of both the mass and C-14 determination. OC and EC concentrations as well as their corresponding fraction of modern for firn and ice samples from Fiescherhorn and Jungfraujoch agree well with published results, validating the new method.

Prospects for measuring the gravitational free-fall of antihydrogen with emulsion detectors

The main goal of the AEgIS experiment at CERN is to test the weak equivalence principle for antimatter. AEgIS will measure the free-fall of an antihydrogen beam traversing a moir'e deflectometer. The goal is to determine the gravitational acceleration with an initial relative accuracy of 1% by using an emulsion detector combined with a silicon μ-strip detector to measure the time of flight. Nuclear emulsions can measure the annihilation vertex of antihydrogen atoms with a precision of ~ 1–2 μm r.m.s. We present here results for emulsion detectors operated in vacuum using low energy antiprotons from the CERN antiproton decelerator. We compare with Monte Carlo simulations, and discuss the impact on the AEgIS project.