Contributors | Affiliation | Role |
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McCarthy, Matthew D. | University of California-Santa Cruz (UCSC) | Principal Investigator |
Ake, Hannah | Woods Hole Oceanographic Institution (WHOI BCO-DMO) | BCO-DMO Data Manager |
N, C compound-specific amino acid isotopes from mussels along CA margin: new approach for paleo-isoscape reconstructions.
Sampling
Mussels were collected in the winter (Dec – Feb) of 2009–2010. Sites were chosen to be approximately evenly distributed along the CA coastline, with ,80 km geographic separation between each
sampling site. Our main goal here was to sample mussels from a wide geographic range across the CCS, although for observing finer scale local or regional variations, a finer-scale sampling strategy would like be required. Typically 5 individual mussels were collected from each site, all between 30–40 mm maximum shell length, which were immediately placed on dry ice until further preparation. The adductor muscle of each individual was dissected for analysis. This tissue was selected because isotopic values in muscle tissue have shown relatively long turnover times; based on past growth data, mussels of this size would be expected to integrate approximately annual variability in suspended food source isotopic values for each location sampled.
The dissected adductor tissue was carefully separated from other tissue types, rinsed with deionized water, refrozen, and then freeze-dried for 48 hrs. Lipids were removed using petroleum ether in a Dionex Accelerated Solvent Extractor (Bannockburn, IL). Finally, in preparation for CSI-AA, composite samples were made from a subset of 13 collection sites. For each location chosen for CSI-AA (based on the bulk d15N record), 160.05 mg of lyophilized tissue was weighed and combined for each individual mussel (n = 5).
Elemental and Bulk Isotopic Analyses
Stable carbon (d13C) and nitrogen (d15N) isotope ratios were determined via elemental analyzer isotope ratio mass spectrometry (EA-IRMS) at the University of California, Santa Cruz, Stable Isotope Laboratory (UCSC-SIL; http://emerald.ucsc.edu/~silab/). Approximately 1 mg of each dry isolated DOM sample was weighed into tin capsules (Costec, 5 x 9 mm) for analysis. EA-IRMS analysis was conducted using a Carlo Erba CHNS-O EA1108-elemental analyzer interfaced via a ConFlo III device with a ThermoFinnigan Delta Plus XP isotope ratio mass spectrometer (Thermo Fisher Scientific). Standards, EA-IRMS protocols, and correction routines followed standard UCSC-SIL protocols. Analytical uncertainties of n=3 replicate measurements of isotopic standards ranged from ± 0.05 to 0.1‰ for both d13C and d15N. Carbon to nitrogen elemental ratios were similarly determined by elemental analysis. The presented ratios are atomic ratios (C/N)a normalized to the mass of C and N, but have been abbreviated as C/N throughout.
Compound-specific amino acid Isotopic 13C Analyses
Individual AA d13C values were measured as trifluoroacetyl isopropyl ester (TFA-IP) AA derivatives, after acid hydrolysis. Samples were hydrolyzed by adding 2.5 mg homogenized composite muscle tissue to 1 ml of 6 N HCl, and heating for 20 h at 110 deg C under nitrogen. After drying, AA isopropyl esters were prepared with a 1:5 mixture of AcCl:2-propanol (110 deg C, 60 min) and then acylated using a 1:3 mixture of di chloro - methane (DCM) and trifluoroacetic anhydride (TFAA) (100 deg C, 15 min). Derivatized AAs were dissolved in DCM to a final ratio of approximately 2.5 mg of original tissue to 250 ul DCM for injection on the gas chromatograph IRMS (GC-IRMS) system.
Isotopic analysis was conducted on a Thermo Trace GC Ultra with inline oxidation and reduction furnaces, coupled to a ThermoFinnigan Delta Plus XP IRMS, equipped with a CTC Analytics autosampler.Derivatives (1 ul) were injected (250 deg C constant temperature) onto an Agilent DB-5 column (50 m × 0.32 mm ID × 0.52 um film thickness), with a He carrier flow rate of 2 ml min−1 (constant flow). Separations were achieved with a 4-ramp oven program: 52 deg C, 2 min hold; ramp 1 = 15 deg C min−1 to 75 deg C, hold for 2 min; ramp 2 = 4 deg C min−1 to 185 deg C, hold for 2 min; ramp 3 = 4 deg C min−1 to 200 deg C; ramp 4 = 30 deg C min−1 to 240 deg C, hold for 5 min.
This method allowed for d13C determination of the following AAs in mussel tissue: non-essential AAs alanine (Ala), aspartic acid + asparagine (Asp), glutamic acid + glutamine (Glu), glycine (Gly), proline (Pro), serine (Ser), and tyrosine Tyr); and essential AAs leucine (Leu), isoleucine (Ile), valine (Val), phenylalanine (Phe), and threonine (Thr). Acid hydrolysis destroys tryptophan and cystine, so these were not detected, and it also deaminates\ asparagine to aspartic acid, and glutamine to glutamic acid. While the abbreviations Glx and Asx\ are sometimes used to denote the combined Gln+Glu and Asn+Asp peaks, in order to correspond better with extant CSI-AA literature, we have elected to simply use Asp and Glu abbreviations, as defined above.
All samples were analyzed on the GC-IRMS in triplicate, and measured AA d13C values were corrected for the C added during derivatization, following the approach of Silfer et al. (1991). Reproducibility for tissue samples was typically less than <0.3‰ (n = 3). The average mean deviation for all tissue sample replicates was 0.4‰.
Compound-specific amino acid Isotopic 15N Analyses
Amino acid d15N values were measured as Trifluoroacetyl isopropyl ester (TFA-IP) AA derivatives, following protocols described in detail elsewhere (e.g.,. Briefly, samples were hydrolyzed (6 N HCl, 20 hr at 110uC) under nitrogen, and TFA derivatives subsequently prepared from free AA using a modified version of the protocol described by Silfer (Silfer et al. 1991): isopropyl esters were made with a 1:5 mixture of Acetyl Chloride (AcCl):2-propanol (110uC, 60 minutes), and then acylated using a 1:3 mixture of Dichloromethane:Trifluroacetyl acetate (DCM:TFAA) (100uC, 15 minutes). Derivatized AAs were dissolved in DCM to a final ratio of approximately 4 mg of original tissue to 250 ml DCM. After derivatization, samples were analyzed by a Varian gaschromatograph coupled to a Finnegan Delta-Plus isotope ratiomass spectrometer (GC-IRMS). AAs were separated using a 50 m, 0.32 ID Hewlett Packard Ultra-1 column with 1 mm film thickness. Under our analytical conditions, d15N values could be reproducibly measured for alanine (Ala), aspartic acid + asparagine (Asp), glutamic acid + glutamine (Glu), leucine (Leu), isoleucine (Ile), proline (Pro), valine (Val), glycine (Gly), lysine (Lys), serine (Ser), phenylalanine (Phe), threonine (Thr), and tyrosine (Tyr) (Fig. S4). Most AAs were measured with a standard error of ,1.0% (based on n = 4 injections), and the average mean deviations for individual AA d15N measurements across all tissue sample replicates was 0.5%.
Thermo-Finnigan Isodat software and Microsoft Excel 2013.
BCO-DMO Processing:
- modified parameter names to conform with BCO-DMO naming conventions;
- replaced missing data/blanks with "nd" ("no data")
File |
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mussel_data.csv (Comma Separated Values (.csv), 9.68 KB) MD5:be12f8b7565891addc577773149b5b0d Primary data file for dataset ID 713831 |
Parameter | Description | Units |
Site | Site where sampling occurred | unitless |
Identifier | Sample ID | unitless |
Habitat_Type | Description of habitat | unitless |
n | Number of samples analyzed | count |
Latitude_degMin | Latitude in degree minutes | Degree minutes |
Longitude_degMin | Longitude in degree minutes | Degree minutes |
lat | Latitude in decimal degrees | Decimal degrees |
lon | Longitude in decimal degress | Decimal degrees |
Bulk_d15N | 15N/14N isotopic ratio | per mil |
Bulk_d15N_stdev | Standard deviation of isotopic ratio | per mil |
Bulk_d13C | 13C/12C isotopic ratio | per mil |
Bulk_d13C_stdev | Standard deviation of isotopic ratio | per mil |
Average_Trophic | Average d15N values of [ Alanine, Aspartic Acid, Glutamic Acid, Leucine, Isoleucine, Proline, Valine ] | per mil |
Average_Source | Average d15N values of [ Glycine, Lysine, Phenylalanine, Serine, Tyrosine ] | per mil |
Trophic_Position | Trophic Position; { [ (d15N Glutamic Acid - d15N Phenylalanine) - 3.4 ] / 7.6 } + 1 | unitless |
Ala | Alanine 15N or 13C value | per mil |
Ala_stdev | Alanine standard deviation | per mil |
Asx | Asn+Asp 15N or 13C peak values | per mil |
Asx_stdev | Asx standard deviation | per mil |
Glx | Gln+Glu 15N or 13C peak values | per mil |
Glx_stdev | Glx standard deviation | per mil |
Gly | Glycine 15N or 13C value | per mil |
Gly_stdev | Glycine standard deviation | per mil |
Ile | Isoleucine 15N or 13 C value | per mil |
Ile_stdev | Isoleucine standard deviation | per mil |
Leu | Leucine 15N or 13C value | per mil |
Leu_stdev | Leucine standard deviation | per mil |
Lys | Lysine 15N or 13C value | per mil |
Lys_stdev | Lysine standard deviation | per mil |
Phe | Phenylalanine 15N or 13C value | per mil |
Phe_stdev | Phenylalanine standard deviation | per mil |
Pro | Proline 15N or 13C value | per mil |
Pro_stdev | Proline standard deviation | per mil |
Ser | Serine 15N or 13C value | per mil |
Ser_stdev | Serine standard deviation | per mil |
Thr | Threonine 15N or 13C value | per mil |
Thr_stdev | Threonine standard deviation | per mil |
Tyr | Tyrosine 15N or 13C value | per mil |
Tyr_stdev | Tyrosine standard deviation | per mil |
Val | Valine 15N or 13C value | per mil |
Val_stdev | Valine standard deviation | per mil |
element | Isotopic element; Nitrogen AA or Carbon AA | unitless |
Dataset-specific Instrument Name | Carlo Erba CHNS-O EA1108-elemental analyzer |
Generic Instrument Name | Elemental Analyzer |
Dataset-specific Description | Interfaced via a ConFlo III device with a ThermoFinnigan Delta Plus XP isotope ratio mass spectrometer (Thermo Fisher Scientific) |
Generic Instrument Description | Instruments that quantify carbon, nitrogen and sometimes other elements by combusting the sample at very high temperature and assaying the resulting gaseous oxides. Usually used for samples including organic material. |
Dataset-specific Instrument Name | Trace GC, interfaced via a ConFlo III and GCC device |
Generic Instrument Name | Gas Chromatograph |
Dataset-specific Description | Used with with a ThermoFinnigan Delta Plus XP isotope ratio mass spectrometer (Thermo Fisher Scientific) |
Generic Instrument Description | Instrument separating gases, volatile substances, or substances dissolved in a volatile solvent by transporting an inert gas through a column packed with a sorbent to a detector for assay. (from SeaDataNet, BODC) |
Dataset-specific Instrument Name | hermoFinnigan Delta Plus XP isotope ratio mass spectrometer (Thermo Fisher Scientific) |
Generic Instrument Name | Isotope-ratio Mass Spectrometer |
Dataset-specific Description | Used with Trace GC, interfaced via a ConFlo III and GCC device |
Generic Instrument Description | The Isotope-ratio Mass Spectrometer is a particular type of mass spectrometer used to measure the relative abundance of isotopes in a given sample (e.g. VG Prism II Isotope Ratio Mass-Spectrometer). |
Website | |
Platform | shoreside Calif_shore |
Start Date | 2009-12-01 |
End Date | 2010-02-28 |
Description | Sampling took place in California coast littoral waters from San Diego to the Oregon border |
The bioavailability of nutrients plays a crucial role in oceanic biological productivity, the carbon cycle, and climate change. The global ocean inventory of nitrogen (N) is determined by the balance of N-fixation (sources) and denitrification (sinks). In this three-year project, a researcher from the University of California, Santa Cruz, will focus on developing compound-specific N isotope (d15N) analysis of amino acids as a new tool for understanding N source and transformation of organic matter in paleo-reservoirs. The offsets in the isotopic ratios of individual amino acid groups may yield information about trophic transfer, heterotrophic microbial reworking, and autotrophic versus heterotrophic sources. By measuring and comparing the bulk and amino acid d15N in size-fractioned samples from plankton tows, sediments traps, and multi-cores in oxic and suboxic depositional environments, the researcher will: (1) Provide a proxy of the d15N of average exported photoautotrophic organic matter; and (2) Provide a new level of detail into sedimentary organic N degradation and preservation.
Broader impacts:
This project will improve understanding of the fundamental underpinnings and behaviors of d15N amino acid patterns and how they behave in contrasting sedimentary environments, while also developing a potential paleoceanographic proxy. Funding will support a graduate student and undergraduate research at the institution. The researcher will also conduct community outreach in the form of a workshop/tutorial on the proxy development.
Funding Source | Award |
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NSF Division of Ocean Sciences (NSF OCE) |