Amino acid-specific stable N isotope composition was determined on samples that were hydrolyzed, derivatized, and analyzed according to Popp et al. (2007) and Hannides et al. (2009). Briefly, size-fractioned zooplankton material and target zooplankton taxa were hydrolyzed using trace metal-grade 6 M HCl and the resulting AAs purified using cation exchange chromatography. The samples were then esterified using 4:1 isopropanol:acetyl chloride and derivatized using 3:1 methylene chloride:trifluoroacetyl anhydride. The resulting trifluoroacetyl and isopropyl ester (TFA) derivatives were purified using chloroform extraction and stored at -20°C for up to 1 month before analysis. This method yielded information for the following AAs: alanine (Ala), glycine (Gly), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tyrosine (Tyr), and valine (Val). During acid hydrolysis asparagine (Asn) is converted to aspartic acid (Asp) and glutamine (Gln) is converted to glutamic acid (Glu), thus we also report information on the combined pools, termed Asx (Asn+Asp) and Glx (Gln+Glu), respectively.
TFA derivatives of AAs were analyzed for stable N isotope composition (d15NAA values) following Hannides et al. (2013). AAs were analyzed using a Thermo Scientific Delta V Plus IRMS interfaced to a trace gas chromatograph (GC) fitted with a 60 m BPx5 capillary column through a GC-C III combustion furnace (980°C), reduction furnace (680°C) and liquid nitrogen cold trap. d15NAA values were measured on 3 – 5 replicate injections with norleucine and aminoadipic acid with known d15N values as internal reference materials co-injected on each run.
A composite source d15NAA value was calculated by a weighted averaging a suite of AAs (e.g., d15NSrc-AA = average of Gly, Lys, Phe, and Ser d15N values). Weighting was based the analytical uncertainty calculated from at least triplicate analysis of each sample.