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Effects of hydrolysis on the δ 13 C values of individual amino acids derived from polypeptides and proteins
Author(s) -
Jim Susan,
Jones Vicky,
Copley Mark S.,
Ambrose Stanley H.,
Evershed Richard P.
Publication year - 2003
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.1177
Subject(s) - chemistry , hydrolysis , amino acid , acid hydrolysis , phenylalanine , isotope ratio mass spectrometry , chromatography , gas chromatography , organic chemistry , biochemistry
This study investigates the effects of hydrolysis on the δ 13 C 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 δ 13 C 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 δ 13 C values were statistically identical to bulk SPAA values for all five standards. The accuracy of IAA δ 13 C values was thus shown to be a function of yield; however, poly phenylalanine demonstrated accurate IAA δ 13 C 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 δ 13 C values calculated from measured IAA δ 13 C 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 δ 13 C values of 12 out of its 18 constituent amino acids were determined. Estimated bulk MBC and ABC δ 13 C values were calculated from constituent amino acid δ 13 C values using mass balance. With the exclusion of three ABC samples, calculated bulk bone collagen δ 13 C values ( δ 13 C BC cal ) were shown to correlate extremely well with measured bone collagen values ( δ 13 C BC mes ) for both modern and ancient samples, where R 2 = 0.91 and 0.84, respectively. Significantly, the variation between calculated and measured bone collagen values (Δ 13 C BC cal ‐BC mes ) exhibited similar ranges for both MBC (from −2.6 to +1.2‰) and ABC (from −2.7 to +2.2‰) 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. Copyright © 2003 John Wiley & Sons, Ltd.