Rapid, compound-specific 13 C and 15 N analysis of amino acids: A - PowerPoint PPT Presentation

Rapid, compound-specific δ 13 C and δ 15 N analysis of amino acids: A chloroformate-based method for biological studies Robert G. Walsh, Shaoneng He, Christopher T. Yarnes

CSIA of Amino Acids: Why bother? • Conventional bulk analysis obscures meaningful isotopic variation at the molecular level • Data from a Tree Swallow ( Tachycineta bicolor ) feather: δ 13 (C‰) δ 15 N(‰) Bulk -22.54 9.76 Alanine -24.74 11.56 Aspartic Acid + Asparagine -23.78 10.67 Glutamic Acid + Glutamine -22.93 10.34 Glycine -15.99 12.08 Isoleucine -26.83 16.17 Leucine -33.22 14.03 Lysine -9.83 4.07 Phenylalanine -31.31 4.13 Proline -23.05 16.42 Threonine -22.23 -10.17 Tyrosine -31.17 2.34 Valine -29.40 16.27

CSIA of Amino Acids: Why bother?

CSIA of Amino Acids: Status quo • All methods involve tradeoffs between precision, scope, and time • General approaches Offline HPLC to separate AAs  EA/IRMS • LC/IRMS with/without derivatization • GC/C/IRMS with derivatization • • Prevailing method to prepare amino acids for GC/C/IRMS is esterification/trifluoroacetylation 2-step, 70-minute derivatization reaction • Multiple drying steps, solvents • Strictly anhydrous conditions required •

Objective • Improve ease and efficiency of CSIA of amino acids without compromising accuracy, precision • Ideal method should: • Generate a sample suitable for both C and N analysis • Be compatible with a wide range of biological materials • Require minimal preparation time and instrument time

The Method, Briefly Derivatization: Hydrolysis: GC/C/IRMS 150 ̊ C, 70 min 1 step, 1 min 30m, 40 min methanol pyridine methyl chloroformate Conventional : Conventional : Conventional : 110 ̊ C, 1440 min 60m, 60 min 2 steps, 70 min

Petr Husek (2006) modestly describes his derivatization method: “Like a big bang, a paper on Amino Acid Derivatization and Analysis in Five Minutes appeared in 1991…A trick? By no means! It was pyridine only, a common base in the reaction medium, that caused the miracle…The reagents constituted an era—to such a degree that it was also said: BC – Before Chloroformates, AD – Advanced Derivatization using chloroformates.”

Valine, derivatized 5 ways oxycarbonylation/ esterification valine esterification acetylation/

Benefits of this approach • Hydrolysis High-temperature, short duration acid hydrolysis • minimizes racemization, preserves some amino acids degraded by long-term hydrolysis (Csapó et al. 1997) • Derivatization Microscale (Chen et al. 2010) • One-step aqueous solution derivatization takes minutes • rather than hours, can be used on biological solutions with free amino acids (e.g., blood, cellular lysates) • GC/C/IRMS Small, polar derivatives elute quickly, minimizing • instrument time

Typical chromatograms, reference mixture • From left to right: Alanine, Valine, Glycine, Isoleucine, Leucine, Norleucine (“X”), Proline, Aspartic Acid, Threonine, Methionine, Phenylalanine, Glutamic Acid, Lysine, Histidine, Tyrosine X N Intensity (mV) X C

Nitrogen chromatograms, biological materials Mahi Mahi muscle Phytoplankton, whole organism Right Whale baleen Nori, thallus

• Northern Rough-winged Swallow feather; labels Typical chromatogram, biological sample Intensity (mV) indicate mole percent abundance of the amino acids Alanine (5%) Valine (8%) Glycine (12%) Isoleucine (4%) Leucine (7%) Proline (12%) Aspartic Acid (7%) Threonine (5%) Serine (12%) Methionine (0.4%) Phenylalanine (2%) Glutamic Acid (8%) Lysine (1%) [Histidine (0.4%)] Tyrosine (1%)

Precision • Average precision for standard ( n = 10 preps) • σ (C/N): ± 1.41/ ± 0.98 • This value is total propagated error for carbon; measurement error is ± 0.63 • Average precision for biological samples of chicken egg, whale baleen, seaweed, with n = 4 preparations per material: • σ (C/N): ± 1.67/ ± 0.88

Accuracy with Reference Mixture Good correlation between GC/C/IRMS values and • EA/IRMS values; amino acids with aliphatic side-chains shown

Accuracy with Ala, Glu Standards • Accurate determinations; no significant difference between EA (dark bars) and GC (light bars) values C N

Caveats & Limitations • Low recovery of some amino acids with polar side- chains (e.g., histidine, serine) • Challenges of running samples of unknown AA abundance “blind” • MCF toxicity • Some additional purification may be necessary on some samples, especially high-cellulose, high-lipid

Case Study: Riparian Food Webs

Case Study: Riparian Food Webs • Do songbirds rely more on emergent aquatic insects or terrestrial insect production? Are they part of the algae- or tracheophyte-based food webs? • Performed discriminant analysis using animals with known aquatic diet (n=20 e.g., fish, crustaceans, bivalves) and known terrestrial diet (n=20, e.g., ungulates, canids, insects) from other studies for training; values of Phe (C & N), Glu (C & N) used • How will birds with empirically known aquatic/terrestrial diets be classified?

Birds with Empirically Known Aquatic/T errestrial Diets Eared Grebe Nuttall’s Woodpecker Aquatic invertebrates, fish Wood-boring insects Belted Kingfisher Bushtit Fish Aphids Marsh Wren California Thrasher Emergent aquatic insects Spiders, insects American Dipper Bullock’s Oriole Aquatic invertebrates Grasshoppers, fruit

Probability of Assignment to Correct Diet Group: Habitat Specialists Eared Grebe Nuttall’s Woodpecker 98.1% Aquatic 88.7% Terrestrial Belted Kingfisher Bushtit 97.9% Aquatic 97.4% Terrestrial Marsh Wren California Thrasher 85.4% Aquatic 96.9% Terrestrial American Dipper Bullock’s Oriole 75.5% Aquatic 97.0% Terrestrial

Case Study: Riparian Food Webs • CSIA-AA data have the potential to resolve aquatic versus terrestrial prey sources, a challenge for bulk C/N analysis • Applied to generalist insectivores (Tree Swallows) to look at resource use of aquatic and terrestrial resources during particular life history stages, in drought years, etc. • Findings match up with ecological expectations, other studies Tree Swallow with Callibaetis mayflies

Conclusions & Future Potential • Analysis of biological solutions with free AAs (blood, cell lysates, urine, etc.) to study glutamine, cysteine, and others typically lost in hydrolysis would be novel • Additional time savings possible (microwave hydrolysis, neutralizing samples with NaOH instead of drying, automating derivatization) • Scaling up sampling—capitalizing on short preparation and run times to analyze more samples with similar effort

Acknowledgments • UC Davis Stable Isotope Facility graduate fellowship support • Biological samples donated from many individuals and institutions methyl chloroformate