New strategies for characterizing ancient proteins using matrix-assisted laser desorption ionization mass spectrometry

Abstract Structural characterization of ancient proteins is confounded by the small quantity of material remaining in fossils, difficulties in purification, and the inability to obtain sequence information by classical Edman degradation. We present a microbore reversed phase high performance liquid chromatography (rpHPLC) method for partial purification of small quantities (picomoles) of the bone protein osteocalcin (OC) and subsequent characterization of this material by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The presence of OC in the modern and ancient samples was suggested by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and radioimmunoassay (RIA). The SDS-PAGE of material isolated from 800 yr BP and 10,000 yr BP bones demonstrates a band consistent with the molecular weight of OC and the RIA indicated OC in concentrations of 0.2 to 450ng/mg of bone for samples between 800 and 53,000 yr BP. In modern samples, we demonstrate the use of MALDI-MS to confirm the molecular weight of intact OC and to sequence OC via peptide mass mapping and a novel derivatization approach with post-source decay analysis. MALDI-MS data for three ancient samples with RIA-confirmed osteocalcin (800 yr BP, 10,000 yr BP and 53,000 yr BP) indicate peaks with a molecular mass within the range of modern OC.

[1]  R. Hedges,et al.  The radiocarbon dating of bone , 1989 .

[2]  D. Walton,et al.  Degradation of intracrystalline proteins and amino acids in fossil brachiopods , 1998 .

[3]  John A. Kanis,et al.  Bone and Mineral Research , 1991 .

[4]  Michael Karas,et al.  Matrix‐assisted laser desorption ionization mass spectrometry , 1991 .

[5]  R. Taylor,et al.  AMS radiocarbon dating of bone osteocalcin , 1990 .

[6]  V. Rosen,et al.  Isolation of the human gene for bone gla protein utilizing mouse and rat cDNA clones. , 1986, The EMBO journal.

[7]  J Allison,et al.  An approach to locate phosphorylation sites in a phosphoprotein: mass mapping by combining specific enzymatic degradation with matrix-assisted laser desorption/ionization mass spectrometry. , 1994, Analytical biochemistry.

[8]  H. Bocherens,et al.  A stable isotope study of fossil mammal remains from the Paglicci cave, Southern Italy. N and C as palaeoenvironmental indicators , 1997 .

[9]  S. Carr,et al.  Primary structure of monkey osteocalcin. , 1982, Biochemistry.

[10]  V. Sarich,et al.  Albumin systematics of the extinct mammoth and Tasmanian wolf , 1981, Nature.

[11]  P. Allison,et al.  Book Reviews: Taphonomy. Releasing the Data Locked in the Fossil Record. , 1991 .

[12]  J B Lian,et al.  Osteocalcin and matrix Gla protein: vitamin K-dependent proteins in bone. , 1989, Physiological reviews.

[13]  G. Muyzer,et al.  Preservation of the bone protein osteocalcin in dinosaurs , 1992 .

[14]  R. Demuth,et al.  Presence of osteocalcin and related higher molecular weight 4-carboxyglutamic acid-containing proteins in developing bone. , 1983, The Journal of biological chemistry.

[15]  J. Lian,et al.  Osteocalcin: isolation, characterization, and detection. , 1984, Methods in enzymology.

[16]  N. Ling,et al.  Isolation and sequence of the vitamin K-dependent protein from human bone. Undercarboxylation of the first glutamic acid residue. , 1980, The Journal of biological chemistry.

[17]  W. Meinschein.  Biogeochemistry of amino acids , 1981 .

[18]  J. Watson,et al.  Protein sequencing by matrix-assisted laser desorption ionization-postsource decay-mass spectrometry analysis of the N-Tris(2,4,6-trimethoxyphenyl)phosphine-acetylated tryptic digests. , 1999, Analytical biochemistry.

[19]  R. Michener,et al.  Stable isotopes in ecology and environmental science , 1995 .

[20]  Charge remote fragmentation of peptides following attachment of a fixed positive charge: A matrix-assisted laser desorption/ionization postsource decay study , 1997 .

[21]  S. Weiner,et al.  Interactions of sea-urchin skeleton macromolecules with growing calcite crystals— a study of intracrystalline proteins , 1988, Nature.

[22]  J. Poser,et al.  Characterization of a gamma-carboxyglutamic acid-containing protein from bone. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[23]  G. Eglinton,et al.  Taphonomy: Releasing the data locked in the fossil record , 1991 .

[24]  M. Goodman,et al.  Proboscidean origins of mastodon and woolly mammoth demonstrated immunologically , 1985, Paleobiology.

[25]  G. Muyzer,et al.  Immunological methods in molecular palaeontology , 1991 .

[26]  S. Macko,et al.  Assessment of trophic structure of Cretaceous communities based on stable nitrogen isotope analyses , 1993 .

[27]  K. Brew,et al.  Macromolecules from Living and Fossil Biominerals Implications for the Establishment of Molecular Phylogenies , 1993 .

[28]  K. Brew,et al.  Macromolecules from Living and Fossil Biominerals , 1993 .

[29]  N. Tuross,et al.  New approaches to the study of disease in archeological New World populations. , 1992, Human biology.

[30]  J. Bada.  Amino Acid Racemization Dating of Fossil Bones , 1985 .

[31]  M. Karas,et al.  Ultraviolet–laser desorption/ionization mass spectrometry of femtomolar amounts of large proteins , 1989 .

[32]  J. Bada,et al.  Decomposition of hydroxy amino acids in foraminiferal tests; kinetics, mechanism and geochronological implications , 1978 .

[33]  G. Rodan,et al.  Characterization of the rat osteocalcin gene: stimulation of promoter activity by 1,25-dihydroxyvitamin D3. , 1988, Biochemistry.

[34]  A. Tseng,et al.  Partial amino acid sequence of osteocalcin from an extinct species of ratite bird. , 1990, Biochemistry international.

[35]  P. Hauschka.  Quantitative determination of γ-carboxyglutamic acid in proteins , 1977 .

[36]  S. Carr,et al.  Gas chromatographic mass spectrometric sequence determination of osteocalcin, a gamma-carboxyglutamic acid-containing protein from chicken bone. , 1981, The Journal of biological chemistry.

[37]  G. Stein,et al.  Structure of the rat osteocalcin gene and regulation of vitamin D-dependent expression. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[38]  Teh Lc,et al.  The amino acid sequences of goat, pig and wallaby osteocalcins. , 1984 .

[39]  L. Stathoplos,et al.  [9] Ancient proteins in fossil bones , 1993 .

[40]  S. Carr,et al.  Calcium-dependent alpha-helical structure in osteocalcin. , 1982, Biochemistry.

[41]  P. Hauschka,et al.  Comparison of bone collagen and osteocalcin for determination of radiocarbon ages and paleodietary reconstruction , 1991 .

[42]  P. E. Hare,et al.  Variability in the preservation of the isotopic composition of collagen from fossil bone , 1988 .

[43]  G. Gonnet,et al.  Protein identification by mass profile fingerprinting. , 1993, Biochemical and biophysical research communications.

[44]  B. Fry,et al.  Sources and Cycling of Organic Matter within Modern and Prehistoric Food Webs , 1993 .

[45]  D. Gage,et al.  Characterization of the malonyl-CoA-sensitive carnitine palmitoyltransferase (CPTo) of a rat heart mitochondrial particle. Evidence that the catalytic unit is CPTi. , 1994, The Journal of biological chemistry.

[46]  J. Watson,et al.  Matrix-assisted laser desorption ionization mass spectrometry: applications in peptide and protein characterization. , 1995, Protein expression and purification.

[47]  H. Taylor,et al.  Stable isotope geochemistry : a tribute to Samuel Epstein , 1991 .

[48]  K. Piez.  Amino Acid Composition of Some Calcified Proteins , 1961, Science.

[49]  J. Watson,et al.  A picomole-scale method for charge derivatization of peptides for sequence analysis by mass spectrometry. , 1997, Analytical chemistry.

[50]  P. Price,et al.  The propeptide of rat bone gamma-carboxyglutamic acid protein shares homology with other vitamin K-dependent protein precursors. , 1985, Proceedings of the National Academy of Sciences of the United States of America.