Rapid, compound-specific δ13C and δ15N analysis of amino acids ...

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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

Transcript of Rapid, compound-specific δ13C and δ15N analysis of amino acids ...

Page 1: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...

Rapid, compound-specific δ13C and δ15N analysis of amino acids: A chloroformate-based method for biological studies

Robert G. Walsh, Shaoneng He, Christopher T. Yarnes

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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: δ13(C‰) δ15N(‰)

Bulk -22.54 9.76Alanine -24.74 11.56Aspartic Acid + Asparagine -23.78 10.67Glutamic Acid + Glutamine -22.93 10.34Glycine -15.99 12.08Isoleucine -26.83 16.17Leucine -33.22 14.03Lysine -9.83 4.07Phenylalanine -31.31 4.13Proline -23.05 16.42Threonine -22.23 -10.17Tyrosine -31.17 2.34Valine -29.40 16.27

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CSIA of Amino Acids: Why bother?

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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

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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

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The Method, Briefly

Hydrolysis: 150̊C, 70 min

Conventional: 110̊C, 1440 min

methyl chloroformate

pyridine

methanol

Derivatization: 1 step, 1 min

Conventional: 2 steps, 70 min

GC/C/IRMS 30m, 40 min

Conventional: 60m, 60 min

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“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.”

Petr Husek (2006) modestly describes his derivatization method:

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oxycarbonylation/ esterification

acetylation/ esterification

valine

Valine, derivatized 5 ways

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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

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• From left to right: Alanine, Valine, Glycine, Isoleucine, Leucine, Norleucine (“X”), Proline, Aspartic Acid, Threonine, Methionine, Phenylalanine, Glutamic Acid, Lysine, Histidine, Tyrosine

Inte

nsity

(mV

)

C

N

X

X

Typical chromatograms, reference mixture

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• Northern Rough-winged Swallow feather; labels indicate mole percent abundance of the amino acids

Alan

ine (5

%)

Glyc

ine (1

2%)

Valin

e (8%

)

Isoleu

cine (

4%)

Leuc

ine (7

%)

Proli

ne (1

2%)

Aspa

rtic A

cid (7

%)

Thre

onine

(5%

) Se

rine (

12%

) Me

thion

ine (0

.4%)

Phen

ylalan

ine (2

%)

Glut

amic

Acid

(8%)

Lysin

e (1%

) [H

istidi

ne (0

.4%)]

Tyro

sine (

1%)

Inte

nsity

(mV

) Typical chromatogram, biological sample

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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

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

Accuracy with Reference Mixture

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C N

• Accurate determinations; no significant difference between EA (dark bars) and GC (light bars) values

Accuracy with Ala, Glu Standards

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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

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Case Study: Riparian Food Webs

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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?

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Birds with Empirically Known Aquatic/Terrestrial 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

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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

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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

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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

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methyl chloroformate

• UC Davis Stable Isotope Facility graduate fellowship support

• Biological samples donated from many individuals and institutions

Acknowledgments