Assessing the Extent of Bone Degradation Using Glutamine Deamidation in Collagen Julie Wilson,* ,†,‡ Nienke L. van Doorn, § and Matthew J. Collins § † Department of Mathematics, University of York, York YO10 5YW, U.K. ‡ Department of Chemistry, University of York, York YO10 5YW, U.K. § BioArCh, Biology (S-Block), University of York, York YO10 5YW, U.K. * S Supporting Information ABSTRACT: Collagen peptides are analyzed using a low-cost, high- throughput method for assessing deamidation using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). For each chosen peptide, the theoretical distribution is calculated and the measured distribution for each sample compared with this to determine the extent of glutamine deamidation. The deamidation of glutamine (Q) to glutamic acid (E) results in a mass shift of +0.984 Da. Thus, from the resolution of our data, the second peak in the isotope distribution for a peptide containing one glutamine residue coincides with the first peak of the isotope distribution for the peptide in which the residue is deamidated. A genetic algorithm is used to determine the extent of deamidation that gives the best fit to the measured distribution. The method can be extended to peptides containing more than one glutamine residue. The extent of protein degradation assessed in this way could be used, for example, to assess the damage of collagen, and screen samples for radiocarbon dating and DNA analysis. T he nonenzymatic deamidation of asparagine (Asn) and glutamine (Gln) residues represents one of the most widely studied post-translational modifications. 1 Asparaginyl deamidation may play a role in timing biological processes and has been implicated in neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. 2,3 Glutamine is generally much more stable than asparagine, with the much slower deamidation rates being attributed to less stable reaction intermediates. 4 As a result, studies relating glutamine deamidation with the aging process involve low-turnover proteins, such as eye lens crystallin, associated with cataract formation. 5 The slower rates of glutaminyl deamidation make it suitable for the dating of historical artifacts. Mass spectrometry has been used to study the deterioration and aging of proteinaceous paint media used in works-of-art to allow suitable restoration processes to be designed. 6 The deamidation of Gln was found to be a major factor in the degradation process, with asparagine deamidation occurring too fast in their samples to be useful. The rate of Asn deamidation is strongly affected by both primary and higher order structure, 7 but it has been shown that the level of asparaginyl deamidation could be useful for dating (structurally coherent) wool textiles from museum collections. 8 Rates of asparagine and glutamine deamidation are heavily influenced by neighboring residues. The analysis of pentapep- tides with different permutations of adjacent amino acids showed that, under physiological conditions (pH 7.4), half-lives for asparagine ranged from 6 to 507 days in comparison to 96 to 3409 days for glutamine in similar positions. 9 Peptides with small charged residues on either side of the amide were found to have the highest rates. By modeling peptide deamidation and interpolating between experimental values, the list of sequence- dependent rates was extended to include half-lives of up to 50 years. 10 Although the three-dimensional structure makes the influence of neighboring groups more complex in proteins than in short peptides, they found that the effects could be estimated where structures were available. Although few protein studies involving glutamine deamidation have been reported due to the slow rates, significant levels were detected in a recombinant monoclonal antibody, suggesting a possible role in the binding affinity of the protein. 11 As expected, glutamine residues at different locations in the three-dimensional structure showed different susceptibilities to deamidation. Type I collagen is the dominant protein in bone and widely used in archeology for radiocarbon ( 14 C) and stable isotope analyses. 12 The deamidation of collagen for dating materials has been discussed by Hurtado and O’Connor. 14 The upper-limit of radiocarbon dating even in the best-preserved bones is 60 000 years; however, collagen can persist for more than 1 order of magnitude longer in temperate environments. 13 Bone persists over the periods in a ‘recrystallization window’ of between pH 7.6 and 8.1, 15 which will buffer the within-bone pH while at the same time destroying the original bone bioapatite. Below pH 7.6, bone will undergo sacrificial dissolution, 11,16 and above pH 8.2 the mineral will persist. Observed rates of glutamine deamidation in the range typical Received: May 21, 2012 Accepted: October 2, 2012 Published: October 2, 2012 Article pubs.acs.org/ac © 2012 American Chemical Society 9041 dx.doi.org/10.1021/ac301333t | Anal. Chem. 2012, 84, 9041-9048