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

Rapid, compound-specific δ13C and δ15N analysis

• f 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:

δ13(C‰) δ15N(‰) 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

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

“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:

• xycarbonylation/

esterification

acetylation/ esterification

valine

Valine, derivatized 5 ways

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

• From left to right: Alanine, Valine, Glycine, Isoleucine, Leucine,

Norleucine (“X”), Proline, Aspartic Acid, Threonine, Methionine, Phenylalanine, Glutamic Acid, Lysine, Histidine, Tyrosine

Intensity (mV) C N

X X

Typical chromatograms, reference mixture

Nitrogen chromatograms, biological materials

Mahi Mahi muscle Phytoplankton, whole organism Nori, thallus Right Whale baleen

• Northern Rough-winged Swallow feather; labels

indicate mole percent abundance of the amino acids

Alanine (5%) Glycine (12%) Valine (8%) 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%)

Intensity (mV)

Typical chromatogram, biological sample

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

• Good correlation between GC/C/IRMS values and

EA/IRMS values; amino acids with aliphatic side-chains shown

Accuracy with Reference Mixture

C N

• Accurate determinations; no significant difference

between EA (dark bars) and GC (light bars) values

Accuracy with Ala, Glu Standards

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
• r 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,

• ther 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

• f drying, automating derivatization)
• Scaling up sampling—capitalizing on short

preparation and run times to analyze more samples with similar effort

methyl chloroformate

• UC Davis Stable Isotope Facility graduate

fellowship support

• Biological samples donated from many individuals

and institutions

Acknowledgments