Bone protein “extractomics”: comparing the efficiency of bone protein extractions of Gallus gallus in tandem mass spectrometry, with an eye towards paleoproteomics
Proteomic studies of bone require specialized extraction protocols to demineralize and solubilize proteins from within the bone matrix. Although various protocols exist for bone protein recovery, little is known about how discrete steps in each protocol affect the subset of the bone proteome recover...
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PeerJ Inc.
2016-10-01
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| author | Elena R. Schroeter Caroline J. DeHart Mary H. Schweitzer Paul M. Thomas Neil L. Kelleher |
| author_facet | Elena R. Schroeter Caroline J. DeHart Mary H. Schweitzer Paul M. Thomas Neil L. Kelleher |
| author_sort | Elena R. Schroeter |
| collection | DOAJ |
| description | Proteomic studies of bone require specialized extraction protocols to demineralize and solubilize proteins from within the bone matrix. Although various protocols exist for bone protein recovery, little is known about how discrete steps in each protocol affect the subset of the bone proteome recovered by mass spectrometry (MS) analyses. Characterizing these different “extractomes” will provide critical data for development of novel and more efficient protein extraction methodologies for fossils. Here, we analyze 22 unique sub-extractions of chicken bone and directly compare individual extraction components for their total protein yield and diversity and coverage of bone proteins identified by MS. We extracted proteins using different combinations and ratios of demineralizing reagents, protein-solubilizing reagents, and post-extraction buffer removal methods, then evaluated tryptic digests from 20 µg aliquots of each fraction by tandem MS/MS on a 12T FT-ICR mass spectrometer. We compared total numbers of peptide spectral matches, peptides, and proteins identified from each fraction, the redundancy of protein identifications between discrete steps of extraction methods, and the sequence coverage obtained for select, abundant proteins. Although both alpha chains of collagen I (the most abundant protein in bone) were found in all fractions, other collagenous and non-collagenous proteins (e.g., apolipoprotein, osteonectin, hemoglobin) were differentially identified. We found that when a standardized amount of extracted proteins was analyzed, extraction steps that yielded the most protein (by weight) from bone were often not the ones that produced the greatest diversity of bone proteins, or the highest degree of protein coverage. Generally, the highest degrees of diversity and coverage were obtained from demineralization fractions, and the proteins found in the subsequent solubilization fractions were highly redundant with those in the previous fraction. Based on these data, we identify future directions and parameters to consider (e.g., proteins targeted, amount of sample required) when applying discrete parts of these protocols to fossils. |
| first_indexed | 2024-03-09T07:01:21Z |
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| last_indexed | 2024-03-09T07:01:21Z |
| publishDate | 2016-10-01 |
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| spelling | doaj.art-ae051db8a1174f6ea84ab322c69ddf9a2023-12-03T09:51:31ZengPeerJ Inc.PeerJ2167-83592016-10-014e260310.7717/peerj.2603Bone protein “extractomics”: comparing the efficiency of bone protein extractions of Gallus gallus in tandem mass spectrometry, with an eye towards paleoproteomicsElena R. Schroeter0Caroline J. DeHart1Mary H. Schweitzer2Paul M. Thomas3Neil L. Kelleher4Department of Biological Sciences, North Carolina State University, Raleigh, NC, United StatesProteomics Center of Excellence and Departments of Chemistry, Molecular Biosciences, and the Feinberg School of Medicine, Northwestern University, Evanston, IL, United StatesDepartment of Biological Sciences, North Carolina State University, Raleigh, NC, United StatesProteomics Center of Excellence and Departments of Chemistry, Molecular Biosciences, and the Feinberg School of Medicine, Northwestern University, Evanston, IL, United StatesProteomics Center of Excellence and Departments of Chemistry, Molecular Biosciences, and the Feinberg School of Medicine, Northwestern University, Evanston, IL, United StatesProteomic studies of bone require specialized extraction protocols to demineralize and solubilize proteins from within the bone matrix. Although various protocols exist for bone protein recovery, little is known about how discrete steps in each protocol affect the subset of the bone proteome recovered by mass spectrometry (MS) analyses. Characterizing these different “extractomes” will provide critical data for development of novel and more efficient protein extraction methodologies for fossils. Here, we analyze 22 unique sub-extractions of chicken bone and directly compare individual extraction components for their total protein yield and diversity and coverage of bone proteins identified by MS. We extracted proteins using different combinations and ratios of demineralizing reagents, protein-solubilizing reagents, and post-extraction buffer removal methods, then evaluated tryptic digests from 20 µg aliquots of each fraction by tandem MS/MS on a 12T FT-ICR mass spectrometer. We compared total numbers of peptide spectral matches, peptides, and proteins identified from each fraction, the redundancy of protein identifications between discrete steps of extraction methods, and the sequence coverage obtained for select, abundant proteins. Although both alpha chains of collagen I (the most abundant protein in bone) were found in all fractions, other collagenous and non-collagenous proteins (e.g., apolipoprotein, osteonectin, hemoglobin) were differentially identified. We found that when a standardized amount of extracted proteins was analyzed, extraction steps that yielded the most protein (by weight) from bone were often not the ones that produced the greatest diversity of bone proteins, or the highest degree of protein coverage. Generally, the highest degrees of diversity and coverage were obtained from demineralization fractions, and the proteins found in the subsequent solubilization fractions were highly redundant with those in the previous fraction. Based on these data, we identify future directions and parameters to consider (e.g., proteins targeted, amount of sample required) when applying discrete parts of these protocols to fossils.https://peerj.com/articles/2603.pdfBone matrix proteinsBone protein extraction protocolsMethods comparisonPaleoproteomicsMass spectrometryProteomics |
| spellingShingle | Elena R. Schroeter Caroline J. DeHart Mary H. Schweitzer Paul M. Thomas Neil L. Kelleher Bone protein “extractomics”: comparing the efficiency of bone protein extractions of Gallus gallus in tandem mass spectrometry, with an eye towards paleoproteomics PeerJ Bone matrix proteins Bone protein extraction protocols Methods comparison Paleoproteomics Mass spectrometry Proteomics |
| title | Bone protein “extractomics”: comparing the efficiency of bone protein extractions of Gallus gallus in tandem mass spectrometry, with an eye towards paleoproteomics |
| title_full | Bone protein “extractomics”: comparing the efficiency of bone protein extractions of Gallus gallus in tandem mass spectrometry, with an eye towards paleoproteomics |
| title_fullStr | Bone protein “extractomics”: comparing the efficiency of bone protein extractions of Gallus gallus in tandem mass spectrometry, with an eye towards paleoproteomics |
| title_full_unstemmed | Bone protein “extractomics”: comparing the efficiency of bone protein extractions of Gallus gallus in tandem mass spectrometry, with an eye towards paleoproteomics |
| title_short | Bone protein “extractomics”: comparing the efficiency of bone protein extractions of Gallus gallus in tandem mass spectrometry, with an eye towards paleoproteomics |
| title_sort | bone protein extractomics comparing the efficiency of bone protein extractions of gallus gallus in tandem mass spectrometry with an eye towards paleoproteomics |
| topic | Bone matrix proteins Bone protein extraction protocols Methods comparison Paleoproteomics Mass spectrometry Proteomics |
| url | https://peerj.com/articles/2603.pdf |
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