Bone protein “extractomics”: comparing the efficiency of bone protein extractions ofGallus gallusin tandem mass spectrometry, with an eye towards paleoproteomics

Abstract

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.

A guide to ancient protein studies

“…A number of protocols have been reported for the extraction of ancient proteins, particularly for the extraction of bone protein [81][82][83] , and include protocols based on SDS buffers and polyacrylamide gels ( 24,84 ), Filter-Aided Sample Preparation (FASP) 1,85,86 , and Gel-Aided Sample Preparation (GASP) 87,88 . However, the efficacy of these protocols, their downstream effect on protein identification and resulting chemically-induced modification have not been systematically compared in studies of ancient proteins, although examples exist that compare their performance on modern material 89,90 .…”

Proteomic method to extract, concentrate, digest and enrich peptides from fossils with coloured (humic) substances for mass spectrometry analyses

“…Although the ‘NaOH’ fraction possessed a greater number of collagen I PSMs than the NaOH-400/200/4 fraction (table 1), the number of unique peptides and per cent sequence coverage for all proteins were either substantially greater in the NaOH-400/200/4 fraction, or roughly equal between the two (electronic supplementary material, table S2). This suggests that unlike demineralization treatments [15], the NaOH pretreatment is not extracting a different subset of proteomic information than the following solubilization step. Further, the nearly identical results obtained from the pretreated (NaOH-400/200/4) and non-treated (400/200/4) fractions (table 1 and electronic supplementary material, table S2) indicate that NaOH pretreatment under these conditions does not improve the capacity of the 400/200/4 to solubilize proteins from the bone matrix.…”

Palaeoproteomic identification of breast milk protein residues from the archaeological skeletal remains of a neonatal dog

“…A total of 143 protein groups (71.5%) were detected in both the EDTA and the pellet fractions, and 154 protein groups (77.0%) were retrieved from both rib and vertebra. Recovered proteomes can differ in multiple experimental fractions extracted from a single sample 32 and from different bone elements from a single individual 33 . PANTHER analysis indicated that 51.6% of protein groups are classified as an extracellular component (Supplementary Table 3) and 41.3% are related with binding function (Supplementary Table 4).…”