Rapid, compound-specific δ13C and δ15N analysis of amino acids ...
Transcript of Rapid, compound-specific δ13C and δ15N analysis of amino acids ...
![Page 1: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/1.jpg)
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
![Page 2: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/2.jpg)
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
![Page 3: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/3.jpg)
CSIA of Amino Acids: Why bother?
![Page 4: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/4.jpg)
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
![Page 5: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/5.jpg)
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
![Page 6: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/6.jpg)
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
![Page 7: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/7.jpg)
“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:
![Page 8: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/8.jpg)
oxycarbonylation/ esterification
acetylation/ esterification
valine
Valine, derivatized 5 ways
![Page 9: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/9.jpg)
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
![Page 10: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/10.jpg)
• 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
![Page 11: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/11.jpg)
Nitrogen chromatograms, biological materials
Mahi Mahi muscle Phytoplankton, whole organism
Nori, thallus Right Whale baleen
![Page 12: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/12.jpg)
• 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
![Page 13: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/13.jpg)
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
![Page 14: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/14.jpg)
• Good correlation between GC/C/IRMS values and EA/IRMS values; amino acids with aliphatic side-chains shown
Accuracy with Reference Mixture
![Page 15: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/15.jpg)
C N
• Accurate determinations; no significant difference between EA (dark bars) and GC (light bars) values
Accuracy with Ala, Glu Standards
![Page 16: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/16.jpg)
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
![Page 17: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/17.jpg)
Case Study: Riparian Food Webs
![Page 18: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/18.jpg)
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?
![Page 19: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/19.jpg)
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
![Page 20: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/20.jpg)
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
![Page 21: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/21.jpg)
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
![Page 22: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/22.jpg)
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
![Page 23: Rapid, compound-specific δ13C and δ15N analysis of amino acids ...](https://reader031.fdocument.org/reader031/viewer/2022021500/588dab081a28ab26218bb52b/html5/thumbnails/23.jpg)
methyl chloroformate
• UC Davis Stable Isotope Facility graduate fellowship support
• Biological samples donated from many individuals and institutions
Acknowledgments