Multiplexed quantitative proteomics using differential metabolic 15N-labeling

There are several advantages of using metabolic labeling in quantitative proteomics. The early pooling of samples compared to post-labeling methods eliminates errors from different sample processing, protein extraction and enzymatic digestion. Metabolic labeling is also highly efficient and relatively inexpensive compared to commercial labeling reagents. However, methods for multiplexed quantitation in the MS-domain (or ‘non-isobaric’ methods), suffer from signal dilution at higher degrees of multiplexing, as the MS/MS signal for peptide identification is lower given the same amount of peptide loaded onto the column or injected into the mass spectrometer. This may partly be overcome by mixing the samples at non-uniform ratios, for instance by increasing the fraction of unlabeled proteins. We have developed an algorithm for arbitrary degrees of nonisobaric multiplexing for relative protein abundance measurements. We have used metabolic labeling with different levels of 15N, but the algorithm is in principle applicable to any isotope or combination of isotopes. Ion trap mass spectrometers are fast and suitable for LC-MS/MS and peptide identification. However, they cannot resolve overlapping isotopic envelopes from different peptides, which makes them less suitable for MS-based quantitation. Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry is less suitable for LC-MS/MS, but provides the resolving power required to resolve overlapping isotopic envelopes. We therefore combined ion trap LC-MS/MS for peptide identification with FTICR LC-MS for quantitation using chromatographic alignment. We applied the method in a heat shock study in a plant model system (A. thaliana) and compared the results with gene expression data from similar experiments in literature.

Microbial isotopic fractionation of perchlorate chlorine

Perchlorate contamination can be microbially respired to innocuous chloride and thus can be treated effectively. However, monitoring a bioremediative strategy is often difficult due to the complexities of environmental samples. Here we demonstrate that microbial respiration of perchlorate results in a significant fractionation (similar to - 15parts per thousand) of the chlorine stable isotope composition of perchlorate. This can be used to quantify the extent of biotic degradation and to separate biotic from abiotic attenuation of this contaminant.

A cross-calibration of chlorine isotopic measurements and suitability of seawater as the international reference material

A collection of 24 seawaters from various worldwide locations and differing depth was culled to measure their chlorine isotopic composition (delta(37)Cl). These samples cover all the oceans and large seas: Atlantic, Pacific, Indian and Antarctic oceans, Mediterranean and Red seas. This collection includes nine seawaters from three depth profiles down to 4560 mbsl. The standard deviation (2sigma) of the delta(37)Cl of this collection is +/-0.08 parts per thousand, which is in fact as large as our precision of measurement ( +/- 0.10 parts per thousand). Thus, within error, oceanic waters seem to be an homogeneous reservoir. According to our results, any seawater could be representative of Standard Mean Ocean Chloride (SMOC) and could be used as a reference standard. An extended international cross-calibration over a large range of delta(37)Cl has been completed. For this purpose, geological fluid samples of various chemical compositions and a manufactured CH3Cl gas sample, with delta(37)Cl from about -6 parts per thousand to +6 parts per thousand have been compared. Data were collected by gas source isotope ratio mass spectrometry (IRMS) at the Paris, Reading and Utrecht laboratories and by thermal ionization mass spectrometry (TIMS) at the Leeds laboratory. Comparison of IRMS values over the range -5.3 parts per thousand to +1.4 parts per thousand plots on the Y=X line, showing a very good agreement between the three laboratories. On 11 samples, the trend line between Paris and Reading Universities is: delta(37)Cl(Reading)= (1.007 +/- 0.009)delta(37)Cl(Paris) - (0.040 +/- 0.025), with a correlation coefficient: R-2 = 0.999. TIMS values from Leeds University have been compared to IRMS values from Paris University over the range -3.0 parts per thousand to +6.0 parts per thousand. On six samples, the agreement between these two laboratories, using different techniques is good: delta(37)Cl(Leeds)=(1.052 +/- 0.038)delta(37)Cl(Paris) + (0.058 +/- 0.099), with a correlation coefficient: R-2 = 0.995. The present study completes a previous cross-calibration between the Leeds and Reading laboratories to compare TIMS and IRMS results (Anal. Chem. 72 (2000) 2261). Both studies allow a comparison of IRMS and TIMS techniques between delta(37)Cl values from -4.4 parts per thousand to +6.0 parts per thousand and show a good agreement: delta(37)Cl(TIMS)=(1.039 +/- 0.023)delta(37)Cl(IRMS)+(0.059 +/- 0.056), with a correlation coefficient: R-2 = 0.996. Our study shows that, for fluid samples, if chlorine isotopic compositions are near 0 parts per thousand, their measurements either by IRMS or TIMS will give comparable results within less than +/- 0.10 parts per thousand, while for delta(37)Cl values as far as 10 parts per thousand (either positive or negative) from SMOC, both techniques will agree within less than +/- 0.30 parts per thousand. (C) 2004 Elsevier B.V. All rights reserved.

Seasonal trends in the stable isotopic composition of snow and meltwater runoff in a subarctic catchment at Okstindan, Norway

Seasonal variations in the stable isotopic composition of snow and meltwater were investigated in a sub-arctic, mountainous, but non-glacial, catchment at Okstindan in northern Norway based on analyses of delta(18)O and deltaD. Samples were collected during four field periods (August 1998; April 1999; June 1999 and August 1999) at three sites lying on an altitudinal transect (740-970 m a.s.l.). Snowpack data display an increase in the mean values of delta(18)O (increasing from a mean value of - 13.51 to - 11.49% between April and August), as well as a decrease in variability through the melt period. Comparison with a regional meteoric water line indicates that the slope of the delta(18)O - deltaD line for the snowpacks decreases over the same period, dropping from 7.49 to approximately 6.2. This change points to the role of evaporation in snowpack ablation and is confirmed by the vertical profile of deuterium excess. Snowpack seepage data, although limited, also suggest reduced values of deltaD, as might be associated with local evaporation during meltwater generation. In general, meltwaters were depleted in delta(18)O relative to the source snowpack at the peak of the melt (June), but later in the year (August) the difference between the two was not statistically significant. The diurnal pattern of isotopic composition indicates that the most depleted meltwaters coincide with the peak in temperature and, hence, meltwater production.

Diet and diversity at later medieval Fishergate: The isotopic evidence

We present the results of stable carbon and nitrogen isotope analysis of bone collagen for 155 individuals buried at the Later Medieval (13th to early 16th century AD) Gilbertine priory of St. Andrew, Fishergate in the city of York (UK). The data show significant variation in the consumption of marine foods between males and females as well as between individuals buried in different areas of the priory. Specifically, individuals from the crossing of the church and the cloister garth had consumed significantly less marine protein than those from other locations. Isotope data for four individuals diagnosed with diffuse idiopathic skeletal hyperostosis (DISH) are consistent with a diet rich in animal protein. We also observe that isotopic signals of individuals with perimortem sharp force trauma are unusual in the context of the Fishergate dataset. We discuss possible explanations for these patterns and suggest that there may have been a specialist hospital or a local tradition of burying victims of violent conflict at the priory. The results demonstrate how the integration of archaeological, osteological, and isotopic data can provide novel information about Medieval burial and society.

Consumer welfare effects of introducing and labeling genetically modified food

Non-hypothetical valuations obtained from experimental auctions in three United States and two European locations were used to calculate welfare effects of introducing and labeling of genetically modified food. Under certain assumptions, we find that introduction of genetically modified food has been welfare enhancing, on average, for United States consumers but not so for Europeans and while mandatory labeling has been beneficial for European consumers, such a policy would be detrimental in the United States. (c) 2005 Elsevier B.V. All rights reserved.

Heat-shock response in Arabidopsis thaliana explored by multiplexed quantitative proteomics using differential metabolic labeling

We have developed a general method for multiplexed quantitative proteomics using differential metabolic stable isotope labeling and mass spectrometry. The method was successfully used to study the dynamics of heat-shock response in Arabidopsis thaliana. A number of known heat-shock proteins were confirmed, and some proteins not previously associated with heat shock were discovered. The method is applicable in stable isotope labeling and allows for high degrees of multiplexing.

Quantitative proteomics using uniform 15N-labeling, mascot and the trans-proteomic pipeline

Stable isotope labeling combined with MS is a powerful method for measuring relative protein abundances, for instance, by differential metabolic labeling of some or all amino acids with 14N and 15N in cell culture or hydroponic media. These and most other types of quantitative proteomics experiments using high-throughput technologies, such as LC-MS/MS, generate large amounts of raw MS data. This data needs to be processed efficiently and automatically, from the mass spectrometer to statistically evaluated protein identifications and abundance ratios. This paper describes in detail an approach to the automated analysis of uniformly 14N/15N-labeled proteins using MASCOT peptide identification in conjunction with the trans-proteomic pipeline (TPP) and a few scripts to integrate the analysis workflow. Two large proteomic datasets from uniformly labeled Arabidopsis thaliana were used to illustrate the analysis pipeline. The pipeline can be fully automated and uses only common or freely available software.

Quantitative proteomics using uniform N-15-labeling, MASCOT, and the trans-proteomic pipeline

Stable isotope labeling combined with MS is a powerful method for measuring relative protein abundances, for instance, by differential metabolic labeling of some or all amino acids with N-14 and N-15 in cell culture or hydroponic media. These and most other types of quantitative proteomics experiments using high-throughput technologies, such as LC-MS/MS, generate large amounts of raw MS data. This data needs to be processed efficiently and automatically, from the mass spectrometer to statistically evaluated protein identifications and abundance ratios. This paper describes in detail an approach to the automated analysis of Uniformly N-14/N-15-labeled proteins using MASCOT peptide identification in conjunction with the trans-proteomic pipeline (TPP) and a few scripts to integrate the analysis workflow. Two large proteomic datasets from uniformly labeled Arabidopsis thaliana were used to illustrate the analysis pipeline. The pipeline can be fully automated and uses only common or freely available software.

Hydroponic isotope labeling of entire plants (HILEP) for quantitative plant proteomics

Quantitative analysis by mass spectrometry (MS) is a major challenge in proteomics as the correlation between analyte concentration and signal intensity is often poor due to varying ionisation efficiencies in the presence of molecular competitors. However, relative quantitation methods that utilise differential stable isotope labelling and mass spectrometric detection are available. Many drawbacks inherent to chemical labelling methods (ICAT, iTRAQ) can be overcome by metabolic labelling with amino acids containing stable isotopes (e.g. 13C and/or 15N) in methods such as Stable Isotope Labelling with Amino acids in Cell culture (SILAC). SILAC has also been used for labelling of proteins in plant cell cultures (1) but is not suitable for whole plant labelling. Plants are usually autotrophic (fixing carbon from atmospheric CO2) and, thus, labelling with carbon isotopes becomes impractical. In addition, SILAC is expensive. Recently, Arabidopsis cell cultures were labelled with 15N in a medium containing nitrate as sole nitrogen source. This was shown to be suitable for quantifying proteins and nitrogen-containing metabolites from this cell culture (2,3). Labelling whole plants, however, offers the advantage of studying quantitatively the response to stimulation or disease of a whole multicellular organism or multi-organism systems at the molecular level. Furthermore, plant metabolism enables the use of inexpensive labelling media without introducing additional stress to the organism. And finally, hydroponics is ideal to undertake metabolic labelling under extremely well-controlled conditions. We demonstrate the suitability of metabolic 15N hydroponic isotope labelling of entire plants (HILEP) for relative quantitative proteomic analysis by mass spectrometry. To evaluate this methodology, Arabidopsis plants were grown hydroponically in 14N and 15N media and subjected to oxidative stress.

Cosmopolitan Catterick? Isotopic evidence for population mobility on Rome’s Northern frontier

In order to investigate how the population diversity at major Romano-British urban centres compared to small towns and military outposts, we conducted multi-isotope (carbon, nitrogen, oxygen and strontium) analyses of bones (42 individuals) and teeth (26 individuals) of human skeletons from Cataractonium/ Roman Catterick in North Yorkshire (U.K.). The results suggest a markedly less diverse population at Catterick than at the larger towns. Significant differences are observed between burials from the town and fort area and the suburb of Bainesse to the south, and it is suggested that these reflect a shift to more localised recruitment for the Roman army in the Late Roman period. Isotope data for the ‘Bainesse Eunuch’, an unusual 4th century burial that has been interpreted as the remains of a ‘transvestite’ priest of Cybele, are not ultimately conclusive but consistent with origins in Southern Britain or areas with a similar climate abroad. This paper also presents strontium isotope data for modern vegetation samples from 17 sites in the Catterick/northern Vale of York area which contribute to a continuing effort to map the biosphere 87Sr/86Sr variation in Britain.