Protein coverage for markers of major bone cell types with tryptic peptides in the sequence shown in green.

Q05117: Tartrate-resistant acid phosphatase type 5 is a common marker to identify Osteoclasts showing a 42% peptide coverage. P07214: SPARC is a common marker to identify Osteoblasts showing a 51% peptide coverage. P70669: Phosphate-regulating neutral endopeptidase (PHEX) a common marker to identify Osteocytes showing a 39% peptide coverage. Q99P68: Sclerostin (SOST) a common marker to identify Osteocytes showing a 14% coverage. Q9EPC2: Fibroblast Growth Factor 23 (FGF23) a common marker to identify Osteocytes showing a 7% coverage. (PPTX)</p

S4 Table -

A) All 1633 identified proteins from the Female vs Male comparison of mouse femurs with ≥2 unique peptides. B) All 283 significantly changing proteins in the comparison with q2FC > 0.58. (XLSX)</p

Analysis of hydroxyproline modifications on collagen I alpha chain I.

Two tryptic peptides of collagen 1 alpha chain 1 (COL1A1) with highly-confident hydroxyproline site localizations due to specific and comprehensive fragmentation. A) Representative spectrum displaying the ion series for GDTGA704PoxGA707PoxGSQGA713PoxGLQGM719PoxGER. Fragmentation series provides site localization evidence to confirm modified prolines based on direct and indirect ion evidence including the y4, y5, y10, y16, and y19 ions. B) Extracted ion chromatograms (XIC) of GDTGA704PoxGA707PoxGSQGA713PoxGLQGM719PoxGER precursor ion (m/z = 743.67, z = 3+) on the left and fragment ions (y10, y4, and y19) on the right. C) Similar representative spectrum displaying the fragment ions for the singly modified GLTGS533PoxGSPGPDGK. Direct ion evidence for the modified peptide can be attributed to the y4, y6, and y9 ions. D) XIC of GLTGS 533PoxGSPGPDGK precursor ion (m/z = 621.80 and z = 2+) on the left and fragment ions (y9, y6, and y10) on the right.</p

S1 Table -

A) Protein report for MS analysis of technical injections of femur lysate showing 712 protein identifications with ≥2 unique peptides. B) Peptide report for MS analysis showing the identification of 13210 precursor ions. (XLSX)</p

Comparison of bone microenvironments in healthy and aged/diseased states.

As bones age and the senescence burden increases, the bone microenvironment changes, which is implicated in bone fragility and in osteoarthritis. Additionally, osteocyte lacunocanalicular networks lose canaliculi, and chondrocyte homeostasis is disrupted, causing cartilage degeneration that is accompanied by chondrocyte hypertrophy and osteophyte (bony spurs) formation.</p

S9 Table -

A) Spectral Library created for hydroxyproline analysis from DDA acquisitions of 5 WT mouse femurs containing 603,147 identified peptides across all 5 injections. (XLSX)</p

Proteomic results and Gene Ontology analysis for five wild-type femurs.

Proteins identified in wild-type mouse femurs were analyzed using ConsensusPathDB-mouse (Release MM11, 14.10.2021) GO for both A) “cellular compartment” and B) “biological processes.” The most enriched cellular compartments were the cytoplasm and nucleus while the most significant GO Biological Process was “collagen fibril organization”. C) Protein sequence coverage for secreted protein acidic and cysteine rich (SPARC), an osteoblast marker, is highlighted where green sections of the sequence (51%) are confidently identified in the proteomics data. D) directDIA generated Pseudo-MS/MS spectrum (constructed from DIA generated spectral library) and extracted ion chromatogram of the SPARC peptide LEAGDHPVELLAR (m/z = 710.38, z = 2+) illustrates peak quality and intensity for 2 selected samples, WT1 and WT3.</p

S8 Table -

A) DIA Analysis scheme describing isolation window size and collision energy. (XLSX)</p

Overview of collagen subtypes identified in five wild-type mouse femurs.

A horizontal bar graph shows the estimated abundance of individual collagen proteins confidently identified in the analysis, for example, fibrillar collagens account for 78% of total collagen abundance, mostly from Collagen type 1. Additionally, collagen structural families are quantified and denoted in panels A-F with their percent contribution of total collagen abundance as represented in the vertical bar graph on the right. A: Fibrillar, B: FACIT’s, C: Hexagonal Networks, D: Networks, E: Beaded Filaments, F: Other.</p

Comparison of five wild-type mouse femurs to the core matrisome database and the Core SASP.

A) Proteins identified from wild-type mouse femurs were compared to components of the core matrisome showing most representation to least representation as follows: Collagens (24 proteins), glycoproteins (71 proteins), proteoglycans (19 proteins), ECM affiliated proteins (24 proteins), ECM regulators (70 proteins), secreted proteins (22 proteins) [64–66]. B) Additional comparisons of the bone proteome to the Core SASP show 131 overlapping protein identifications and only 19 unique core SASP proteins [25].</p