Twentieth-Century Paleoproteomics: Lessons from Venta Micena Fossils

Abstract

Proteomics methods can identify amino acid sequences in fossil proteins, thus making it possible to determine the ascription or proximity of a fossil to other species. Before mass spectrometry was used to study fossil proteins, earlier studies used antibodies to recognize their sequences. Lowenstein and colleagues, at the University of San Francisco, pioneered the identification of fossil proteins with immunological methods. His group, together with Olivares's group at the University of Granada, studied the immunological reactions of proteins from the controversial Orce skull fragment (VM-0), a 1.3-million-year-old fossil found at the Venta Micena site in Orce (Granada province, southern Spain) and initially assigned to a hominin. However, discrepancies regarding the morphological features of the internal face of the fossil raised doubts about this ascription. In this article, we review the immunological analysis of the proteins extracted from VM-0 and other Venta Micena fossils assigned to hominins and to other mammals, and explain how these methods helped to determine the species specificity of these fossils and resolve paleontological controversies.

Keywords: ELISA; RIA; VM-0; VM-1960; Venta Micena site; fossil proteins; paleoproteomics.

Figure 1

Preparation of fossil extracts.

Figure 2

Reactivity of EDTA extracts of fossils from the Venta Micena site with three polyspecific mouse sera against human, baboon, and horse albumin in an enzyme-linked immunosorbent assay. VM-0 (skull fragment) and VM-1960 (humeral diaphysis) were assigned to hominins; EEG is a skull fragment of an equid fossil. All three specimens were dated to 1.3 million years old. The results are expressed as relative immunological similarity, defined as the ratio of each reaction to the homologous (most specific) albumin determination. VM-0 and VM-1960 produced a pattern of reactions similar to modern human albumin, whereas EEG produced a pattern of reactions similar to modern horse albumin. From Borja et al. [42], with permission.

Figure 3

Enzyme-linked immunosorbent assay quantification of albumin in the PBS, EDTA and acetic acid extracts of human fresh bone (a fragment of femoral diaphysis), cadaver bone (a fragment of human occipital buried for approximately 10 years), fossils assigned to hominins (VM-0, VM-1960, CV-1), and soil collected around VM-0. Also shown are the buffers used for extractions, tested in the absence of bone. A mouse anti-human albumin polyclonal antiserum was used in all assays. From Lowenstein et al. [45], with permission.

Figure 4

Enzyme-linked immunosorbent quantification of albumin in EDTA extracts of fossils VM-0 and VM-1960, assigned to hominins, and fossil EEG, assigned to an equid, with monoclonal antibodies. Four monoclonal anti-human albumin antibodies (MEGA-1, MEGA-2, MEGA-3 and 8F6F9) were used with the fossils. Polyclonal mouse anti-human albumin serum was used for comparison. Human albumin (25 ng) was tested for comparison. From Lowenstein et al. [45], with permission.

Figure 5

Differences in reactivity and quantification of fossil proteins (short peptide) in comparison to the native protein in assays with a polyclonal antiserum or a monoclonal antibody. Figure created in BioRender.com.

Figure 6

Quantities of human IgG (blue bars) and horse IgG (red bars) detected in PBS, EDTA and acetic acid extracts of (A) hominin and (B) equid fossils from Venta Micena, determined with quantitative dot-blotting. The extraction solutions were used as negative controls. From Torres et al. [51], with permission.