Pyrolysis-GC×GC-TOFMS as a tool for distinguishing the macromolecular structure of nitrogen-bearing animal biopolymers in fossil tissues

Abstract

With the increasing interest in characterization of biopolymers in fossils and their preservation mechanisms, there has been a concomitant increase in reports of preservation of such macromolecules in organic remains of varying ages. However, there have also been several criticisms of these reports involving N-bearing macromolecules such as difficulty in determining endogeneity, and potential for cross-contamination.We explore the utility of pyrolysis comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry as a technique that could potentially provide reproducible data to address some of those criticisms. We find that the enhanced separation capacity of this technique and its ability to resolve complex compounds in low abundances could potentially allow for more specificity in compound assignments. We attempt to understand its diagnostic efficacy by analysing industry standards of chitin and collagen, and a well-documented fossil melanin from Jurassic squid ink. We find that the distribution of nitrogen-bearing compounds in relation to the non-nitrogenous products in each of the above three biomacromolecules is characteristic: acetamide, acetic acid, levoglucosenone and a suite of N-bearing compounds for standard chitin, N-bearing compounds with a distinct region of substituted cyclic 2,5-diketopiperazines (2,5-DKPs) for standard collagen, and a mixture of N-bearing compounds for fossil melanin. We suggest that 3-acetamidofuran and higher homologues, and 3-acetamidopyrones and higher homologues could be reliable markers for N-acetyl-D-glucosamine, of which chitin is a homomer, and that 2,5-DKPs are reliable markers for proteins – particularly collagen, while conceding that assigning a marker purely based on pyrolysis products is difficult for melanin. We then analyse an Eocene mammal bone to make a preliminary investigation into the viability of this technique. We find that the distribution of pyrolysis products is characteristic and distinguishable even in this fossil material, suggesting that proteins like collagen can potentially be detected even when possibly preserved in a degraded form. We conclude that Py-GC×GC-TOFMS is a promising method yielding reliable data that can be used to distinguish between nitrogen-bearing biomacromolecules, possibly even in fossil tissues, and should be the focus of further analyses.