Age-Related Changes in Post-Translational Modifications of Proteins from Whole Male and Female Skeletal Elements

Abstract

One of the key questions in forensic cases relates to some form of age inference, whether this is how old a crime scene is, when in time a particular crime was committed, or how old the victim was at the time of the crime. These age-related estimations are currently achieved through morphological methods with varying degrees of accuracy. As a result, biomolecular approaches are considered of great interest, with the relative abundances of several protein markers already recognized for their potential forensic significance; however, one of the greatest advantages of proteomic investigations over genomics ones is the wide range of post-translational modifications (PTMs) that make for a complex but highly dynamic resource of information. Here, we explore the abundance of several PTMs including the glycosylation, deamidation, and oxidation of several key proteins (collagen, fetuin A, biglycan, serum albumin, fibronectin and osteopontin) as being of potential value to the development of an age estimation tool worthy of further evaluation in forensic contexts. We find that glycosylations lowered into adulthood but deamidation and oxidation increased in the same age range.

Keywords: biological age estimation; forensic proteomics; post-translational modifications; rat bone.

Figure 1

Number of detected glycosylations of lysine in whole rat proteomes for both male and female rats. Males and females showed similar numbers throughout life, with a peak abundance seen at 6–8 weeks.

Figure 2

Age-related changes in galactosyl (K) and glucosylgalactosyl (K) modifications for both male and female rats. (A) Collagen-I-α-1 with males and females showing similar increase in abundance throughout life. (B) Collagen-I-α-2, showed a much shallower increase in abundance, except for galactosyl (K) in female, which rapidly peaked at 8–10 weeks. (C) Collagen-II-α-1, which steadily decrease in abundance for both male and females.

Figure 3

Abundance of N-linked glycosylations in the overall proteome in both female (A) and male (B) rats. (A) Female rats showed a relatively even but rapid decrease in all N-glycosylations from 3 to 4 weeks old. (B) Males also showed a similar decrease from 3 to 4 weeks, but less rapid than females.

Figure 4

Abundance of O-linked glycosylations in the overall proteome for male and female rats. O-linked glycosylations are present on serine and threonine amino acids. (A,B) serine-related O-linked glycosylations in females and males, respectively, and both showed a decrease throughout life, but females began with an initially higher abundance in Hex(S) than males; and (C,D) threonine-related O-linked glycosylations in females and males, respectively, which showed a steady decline for both sexes.

Figure 5

Graphs showing the proteins that displayed changes in abundance of PTMs across age. (A) Deamidation CO1A2, COBA1, FETUA, PGS1 and ALBU showed such a change. (B) Oxidation of lysine changes showed changes in CO1A1, CO1A2, CO2A1, COBA1, CO5A1, FETUA, PGS1 and OSTP, thus showing the highest number of proteins affected. (C) Oxidation of proline showed changes in CO1A1, CO1A2, COBA1, CO5A1, FETUA, PGS1, FETUA and PGS1. (D) Oxidation of methionine showed changes in CO1A1, COBA1, PGS1 AND ALBU. (E) Arg->GluSA had changes in CO1A1, CO2A1 AND COBA1. (F) Lys->Allysine had changes in CO1A1, CO1A2, CO2A1, COBA1 AND CO5A1. Pro->pyrrolidinone (G) and Pro->pyrrolidone (H) had the same proteins showing age-related changes: CO1A1, CO1A2, CO2A1 and COBA1. Graphs showing PTMs with proteins that showed no age-related change can be found in Supplementary Material Figure S1.