Effects of hydrolysis on the δ¹³C values of individual amino acids derived from polypeptides and proteins

Abstract

This study investigates the effects of hydrolysis on the delta13C values of individual amino acids (IAAs) derived from polypeptide standards, and modern and ancient bone collagen. All IAAs were derivatised to their trifluoroacetyl/isopropyl (TFA/IP) esters for delta13C determination using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Firstly, authentic single poly amino acid standards (SPAAs; n = 5) were hydrolysed for 4, 10, 24 and 48 h. As expected, IAA yields increased as a function of hydrolysis time. Significantly, it was only after 24 h of hydrolysis that IAA delta13C values were statistically identical to bulk SPAA values for all five standards. The accuracy of IAA delta13C values was thus shown to be a function of yield; however, poly phenylalanine demonstrated accurate IAA delta13C values with yields of only 1.4 and 4.3%, after 24 and 48 h of hydrolysis time, respectively. Authentic mixed poly amino acid standards (MPAAs; n = 5) comprising two different amino acids were then hydrolysed for 24 h. Percentage recoveries ranged from 36-95%. Estimates of bulk MPAA delta13C values calculated from measured IAA delta13C values agreed within experimental error with measured bulk MPAA values for three out of the five standards. Finally, the experimental procedure was applied to modern rat (MBCs; n = 20) and ancient ovi-caprine and bovine (ABCs; n = 27) bone collagen samples where the delta13C values of 12 out of its 18 constituent amino acids were determined. Estimated bulk MBC and ABC delta13C values were calculated from constituent amino acid delta13C values using mass balance. With the exclusion of three ABC samples, calculated bulk bone collagen delta13C values (delta13C(BCcal)) were shown to correlate extremely well with measured bone collagen values (delta13C(BCmes)) for both modern and ancient samples, where R2 = 0.91 and 0.84, respectively. Significantly, the variation between calculated and measured bone collagen values (Delta13C(BCcal-BCmes)) exhibited similar ranges for both MBC (from -2.6 to +1.2 per thousand ) and ABC (from -2.7 to +2.2 per thousand ) samples, providing evidence for the preservation of intact collagen in the ancient samples. These results demonstrate that the experimental procedures employed in the acid hydrolytic cleavage of peptides or proteins to their constituent amino acids does not involve significant isotopic fractionation.

Probing dietary change of the Kwädąy Dän Ts'ìnchį individual, an ancient glacier body from British Columbia: II. Deconvoluting whole skin and bone collagen δ13C values via carbon isotope analysis of individual amino acids

“…The split-splitless injector temperature was maintained at 200 C. Co-injected standards of alanine, phenylalanine and lysine TFA-methyl esters (Sigma-Aldrich products T3381, T5006 and T4631) of known isotopic composition were added to each sample immediately prior to GC/C/IRMS analysis to monitor instrument performance. Amino acid d 13 C values and associated errors were calculated from the d 13 C values of their TFA-IP derivatives using correction factors (Docherty et al, 2001;Jim et al, 2003;Silfer et al, 1991). Error propagation was employed to combine both the uncertainty associated with the use of correction factors to account for the kinetic isotope effect and the additional carbon atoms introduced during derivatisation.…”

A novel marine dietary indicator utilising compound-specific bone collagen amino acid δ13C values of ancient humans

“…Amino acid d 13 C values were calculated from the d 13 C values of their TFA/IP derivatives using correction factors [5,14,31].…”

Short- and long-term foraging and foddering strategies of domesticated animals from Qasr Ibrim, Egypt

“…These studies suggest that the essential amino acids are preferentially routed directly from dietary protein. Considerable potential exists for the use of individual collagenous amino acids in the field of palaeodietary reconstruction, however, only a handful of studies have so far determined the d 13 C values of individual amino acids in bone collagen from ancient sources [25][26][27]38,71].…”

“…The solution was blown to dryness under nitrogen and redissolved in 2 mL MeOH and stored at ÿ20 (C until required for analysis. The amino acids were derivatised to their trifluroacetyl(TFA)/ isopropyl(IP) esters using the method described by Macko et al and Jim et al [49,38]. The d 13 C values of the amino acids were determined using a GC-C-IRMS, whilst bulk d 13 C values were determined via continuous flow-IRMS [12].…”