Stable-Isotope Mass Spectroscopy: Quantitation

1. Stable-Isotope Mass Spectroscopy: Quantitation Deby Harris Stuart Hay Jessi Dart

2. Outline What is Isotopic Ratio MS Isotopes and standards Instrumentation Injection/Sample Preparation Acceleration/Separation Detection/Data Quantitative MS Application: Environmental Applications Standards Climate Changes Quantitative MS Application: Proteomics What is proteomics Process of a typical proteomics study Methods to quantify protein levels

3. Isotopic Ratio MS? Requires a dedicated instrument. Sample prep and detection are specific not universal. Can be very expensive. Can only sample one chemical species at a time Sample prep is more extensive than typical MS Environmental Samples (finding missing CO2 sink, effect of warming, etc) Biological-metabolic pathways, drug development Geological-Climate changes over time, sources of minerals Archeology-carbon dating or oxygen dating Food Analysis-natural vs. synthetic pathways

4. Isotopes: Stable isotopes occur in a molar ratio In light elements these can vary significantly. For instance, natural waters exhibit variations of the 2H/1H ratio up to 1:2, and of the 18O/16O ratio up to 1:1.1 These isotopic abundances can be compared to a standard using the equation: dX = (Rsample/Rstandard - 1) x 1000, ‰ where dX is the delta value of the sample for element X (H, O, C, etc.) in parts per thousand (“per mil,” ‰) and R is the molar ratio of the heavy (less common) to light (more common) isotope in the sample and in an international standard

5. Standards: International Atomic Energy Agency National Institute of Standards and Technology The standards are based upon: The Standard Mean Ocean Water (SMOW or VSMOW) or Standard Light Antarctic Precipitation (SLAP) for variations of 2H/1H and 18O/16O in natural water. Peedee Belemnite (PDB) for 18O/16O and 13C/12C for atmospheric CO2 and oceanic carbonate Atmospheric Nitrogen Canyon Diablo Troilite (CDT) for the reference on 34S/32S [Online] [Cited: 12 02, 2010.] http://www.iaea.org/

6. Instrumentation: General Overview

7. Sample Inlet: Continuous vs. Duel Inlet

8. Sample Preparation Continuous flow requires online sample preparation while duel inlet requires off-line. Samples must be converted to gaseous form then combusted then reduced and separated. CO2, N2 and H2O must be generated through oxidative combustion (high temps, CuO or CaO as oxygen sources) Sulfur samples must be converted to SO2 through high temps and excess oxygen Deuterium is most often analyzed through duel inlet. It is important to have special “traps” and pure wires to remove unwanted species . This type of MS will not work if more than one chemical species enters the accelerator.

9. Sample Preparation: TC/EA

10. Ionization/Acceleration/Separation Sample is often condensed or concentrated by a cold finger or other method Ionization can occur by Inductively Coupled Plasma, Thermal Ionization or Electron Impact. The ions are accelerated and separated using a magnet instead of a quadrupole. Sensitivity is more important than resolution. Samples and mass differences are small.

11. Ionization/Acceleration/Separation

12. Detection: Detectors are specific. Not seeking to detect a wide range of masses. Typically only looking for 2 to 3. (m/z 44, 45, 46 for CO2) Most IRMS use an array of Faraday cups but can also use inductively coupled plasma or moving wire. The Faraday cup is a small conductive cup designed to catch charged particles. They are not as sensitive as other detectors (electron multipliers) but are more accurate since there is a direct relationship between measured current and the number of ions.

13. Detection:

14. Data Analysis:

15. Comparison of MS to IRMS Resolution is more important in MS while accuracy and sensitivity are more important in ISMS IRMS is selective to a very limited sample range while MS is fairly universal. IRMS requires rigorous sample preparation IRMS compares the ratio of isotopic abundance to known standards Data analysis is based upon comparison to standards

16. Standards for Measuring Stable Isotopes Hydrogen is measured against Vienna Standard Mean Ocean Water (VSMOW) and Standard Light Antarctic Precipitation (SLAP). 2H/1H ratio is 0.00015575 (VSMOW) 2H/1H ratio is 0.000089089 (SLAP) 18O and 2H values of VSMOW lie close to the upper range of values of naturally occurring O and H isotopes. Levels of the isotopes in SLAP lie at the lower end of the range. SLAP was incorporated as a standard with the intention of normalizing the scales. d2H SLAP/VSMOW = -428% d18O SLAP/VSMOW = -55.5%

17. Standards for Measuring Stable Isotopes Carbon is measured against Vienna Pee Dee Belemnite (VPDB). 13C:12C ratio is 0.0112372

18. Standards for Measuring Stable Isotopes Oxygen is measured against Vienna Standard Mean Ocean Water (VSMOW), Standard Light Antarctic Precipitation (SLAP), and Vienna Pee Dee Belemnite (VPDB). 18O/16O ratio is 0.0020052 (VSMAO) 18O/16O ratio is 0.0018939 (SLAP) 18O/16O ratio is 0.0020672 (VPDB). This standard is generally used for carbonate Oxygen. Nitrogen is measured against atmospheric air. 15N/14N ratio is 0.003676 Sulfur is measures against Canyon Diablo Triolite (CDT). 34S/32S ratio is 0.045005

19. Naturally Occurring Stable Isotopes

20. Stable Isotope Ratio MS All materials where isotope ratios of C, N, O, H, or S are to be determined must be converted to a gas for measurement. Mainly CO2, N2, H2, and SO2 are used. O2, N2O, CO, SF6, and CF4 are also used, but less frequently.

21. Stable Isotope Ratio MS and Global Climate Change Reconstructing past climate is possible by determining trace gas concentrations and stable isotope ratios in samples of air, water, rock, and soils using MS techniques. As climate modeling improves, predictions about the future climate will be made with more confidence. Carbon and Oxygen are exchanged between the atmosphere, the oceans, the terrestrial biosphere, and sediment. Carbon and Oxygen isotopes in atmospheric CO2 provide an effective tool for quantifying the contribution of different components to ecosystem exchange.

22. Stable Isotope Ratio MS and Global Climate Change A sustained rise in atmospheric trace gas concentrations (CO2, N2O, CH4 ) over recent years due to increased energy consumption. Since the isotopic signature of these trace gases can provide information about the origin and fate of the gases, the ability to measure their isotopic signature has become a useful tool in the study of the nature and distribution of sources and sinks of these trace gases.

23. Composition of Isotopic C, O, and H Pools in Terrestrial Ecosystems

24. Stable Isotope Ratio MS and Global Climate Change Photosynthesis pathways selectively retain 18O and 12C, making concentrations of these isotopes higher in plants than in the surrounding atmosphere. Plants discriminate against 13C at the C3 pathway of photosynthesis, therefore plants tend to have less (~-16 to -18 o/oo ) 13C than the CO2 from which it was formed. The Oxygen isotope ratio of atmospheric CO2 is primarily determined by isotope exchange with leaf water, soil water, and surface sea water.

25. Stable Isotope Ratio MS and Global Climate Change Periods of increased global temperatures show a drop in isotopic ratios of both carbon and oxygen. Episodes of lower temperatures are likewise accompanied by increases in d18O and d13C.

26. Stable Isotope Ratio MS and Global Climate Change Ice cores from firn are used to evaluate past air and water compositions. Solid ice is crushed in a vacuum environment. The released gas is cryogenically separated or condensated at 14 K and analyzed. Melted sample water is loaded with CO2, once isotopic equilibrium between the CO2 and the water is reached, the CO2 is analyzed.

27. 27 Quantitative MS Application Proteomics What is proteomics Process of a typical proteomics study Methods to quantify protein levels

28. 28 Proteomics What is proteomics? Proteomics is the large-scale study of proteins particularly their structures and functions. Why is proteomics significant? Life is based on proteins and their interactions. We study the protein characteristics in order to: Understand normal and abnormal cellular processes…identify proteins in a sample as a function of state of the organism or cell under certain conditions. Protein-protein interactions…determine how proteins interact with each other in living organisms. Regulatory networks…identify how and where proteins are modified One practical application is in identifying biomarkers for diseases such as cancer. Sometimes important to measure absolute quantity of protein…but not always. Often just as useful to measure relative abundance between samples.

29. 29 Proteomics Process Protein extraction Cell cultures Tissues Protein purification Fractionation Digestion of combined proteins into peptides Analysis Analysis of peptides using MS-MS Database searching to ID the peptide sequences (and then original proteins) Determination of protein abundance from MS data

30. 30 Quantification Methods

31. 31 Qualitative ProteomicsICAT Methodology

32. 32 Qualitative ProteomicsLabel vs. Label-Free Methodology

33. 33 Quantitative ProteomicsAQUA

34. Outline What is Isotopic Ratio MS Isotopes and standards Instrumentation Injection/Sample Preparation Acceleration/Separation Detection/Data Quantitative MS Application: Environmental Applications Standards Climate Changes Quantitative MS Application: Proteomics What is proteomics Process of a typical proteomics study Methods to quantify protein levels

35. 35 References 1. Stable isotope ratio mass spectrometry in global climate change research. Ghosh, Prosenjit and Brand, Willi A. 2003, s.l. : Internal Journal of Mass Spectrometry, 2003, Vol. 228. 2. ISO Analytical. ISO Analytical. [Online] [Cited: 12 05, 2010.] http://www.iso-analytical.co.uk/ea-irms.html. 3. [Online] [Cited: 12 02, 2010.] http://www.iaea.org/. 4. NIST. [Online] [Cited: 12 02, 2010.] http://www.nist.gov/index.html. 5. Carlson, Char. [Online] [Cited: 12 02, 2010.] community.middlebury.edu/~chem/chemistry/class/.../EA-IRMS.ppt. 6. Bantscheff et al.,”Quantitative mass spectrometry in proteomics: a critical review”, Analytical and Bioanalytical Chemistry, 2007, 389, pp1017-1031. 7. Zhu, et al.,“Mass spectrometry-based label-free quantitative proteomics”, Journal of Biomedicine and Biotechnology, 2010, 840518. 8. Tao, et al., “Advances in quantitative proteomics via stable isotope tagging and mass spectrometry”, Current Opinion in Biotechnology, 2003, 14, pp 110-118. 9. Lau, et al.,”Capture and analysis of quantitative proteomic data”, Journal of Proteomics, 2007, 7, pp 2787-2799. 10. T.B. Coplen. Reporting of Stable Hydrogen, Carbon, and Oxygen Isotopic Abundances. Pure and Appl. Chem. V.66 No.2 (1994) 273-276.