A method for whole protein isolation from human cranial bone

Lyon, Sarah; Mayampurath, Anoop; Rogers, MJ; Wolfgeher, Donald J.; Fisher, Sean M.; Volchenboum, Samuel L.; He, Tong‐Chuan; Reid, Russell R.

doi:10.1016/j.ab.2016.09.021

Cited by 15 publications

(6 citation statements)

References 26 publications

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“…In-gel trypsinization, high-performance liquid chromatography for mass spectrometry and LC/MS-MS data acquisition, and analysis were performed as described previously (38,39) with modifications as detailed in Supplementary Methods.…”

Section: Cell Line Validation and Western Blottingmentioning

confidence: 99%

O-GlcNAcylation Enhances Double-Strand Break Repair, Promotes Cancer Cell Proliferation, and Prevents Therapy-Induced Senescence in Irradiated Tumors

Efimova

Appelbe

Ricco

et al. 2019

Molecular Cancer Research

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The metabolic reprogramming associated with characteristic increases in glucose and glutamine metabolism in advanced cancer is often ascribed to answering a higher demand for metabolic intermediates required for rapid tumor cell growth. Instead, recent discoveries have pointed to an alternative role for glucose and glutamine metabolites as cofactors for chromatin modifiers and other protein posttranslational modification enzymes in cancer cells. Beyond epigenetic mechanisms regulating gene expression, many chromatin modifiers also modulate DNA repair, raising the question whether cancer metabolic reprogramming may mediate resistance to genotoxic therapy and genomic instability. Our prior work had implicated N-acetyl-glucosamine (GlcNAc) formation by the hexosamine biosynthetic pathway (HBP) and resulting protein O-GlcNAcylation as a common means by which increased glucose and glutamine metabolism can drive double-strand break (DSB) repair and resistance to therapyinduced senescence in cancer cells. We have examined the effects of modulating O-GlcNAcylation on the DNA damage response (DDR) in MCF7 human mammary carcinoma in vitro and in xenograft tumors. Proteomic profiling revealed deregulated DDR pathways in cells with altered O-GlcNAcylation. Promoting protein O-GlcNAc modification by targeting O-GlcNAcase or simply treating animals with GlcNAc protected tumor xenografts against radiation. In turn, suppressing protein O-GlcNAcylation by blocking O-GlcNAc transferase activity led to delayed DSB repair, reduced cell proliferation, and increased cell senescence in vivo. Taken together, these findings confirm critical connections between cancer metabolic reprogramming, DDR, and senescence and provide a rationale to evaluate agents targeting O-GlcNAcylation in patients as a means to restore tumor sensitivity to radiotherapy. Implications: The finding that the HBP, via its impact on protein O-GlcNAcylation, is a key determinant of the DDR in cancer provides a mechanistic link between metabolic reprogramming, genomic instability, and therapeutic response and suggests novel therapeutic approaches for tumor radiosensitization.

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Section: Cell Line Validation and Western Blottingmentioning

confidence: 99%

O-GlcNAcylation Enhances Double-Strand Break Repair, Promotes Cancer Cell Proliferation, and Prevents Therapy-Induced Senescence in Irradiated Tumors

Efimova

Appelbe

Ricco

et al. 2019

Molecular Cancer Research

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“…13 Currently, most searches of human-derived bone proteomics data have utilized a single search engine (either Mascot, 8 Andromeda, 14 or Sequest 7 ) and, often, have not reported estimates of the FDR. 6 For example, one of the largest bone protein data sets published by Alves et al 8 identified 3038 unique proteins from femoral bone. However, Mascot scores only were used to classify identifications, rather than a FDR.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Despite comprising a significant proportion of the human body mass, bone remains poorly characterized by proteomics compared to other tissuesmainly due to technical challenges associated with tissue extraction and past use of outdated two-dimensional (2D) gel electrophoresis spot analyses. A variety of methods have been employed to solubilize bone, which in animal models have typically involved the use of demineralization agents to remove the inorganic component and chaotropes to extract organic fractions prior to shotgun LC-MS/MS analysis or, in the past, by 2D gel electrophoresis followed by MS. , For investigation of fresh (i.e., nonarcheological) human bone, these more extensively characterized methods of bone protein extraction have not yet been adapted for proteomics, and instead the limited number of studies conducted by LC-MS/MS have used protein extraction protocols utilizing detergents, , or Trizol reagent, which has resulted in low numbers of protein identifications: For example, an analysis of human alveolar bone by Salmon et al reported just 423 protein identifications at less than 1% false discovery rate (FDR).…”

Section: Introductionmentioning

confidence: 99%

“…Despite significant overlap, different search engines will preferentially identify different subsets of peptides; therefore, the searching of data with multiple algorithms and combination of results using software such as iProphet can increase confidence in peptide assignment . Currently, most searches of human-derived bone proteomics data have utilized a single search engine (either Mascot, Andromeda, or Sequest) and, often, have not reported estimates of the FDR . For example, one of the largest bone protein data sets published by Alves et al identified 3038 unique proteins from femoral bone.…”

Section: Introductionmentioning

confidence: 99%

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Proteomic and N-Terminomic TAILS Analyses of Human Alveolar Bone Proteins: Improved Protein Extraction Methodology and LysargiNase Digestion Strategies Increase Proteome Coverage and Missing Protein Identification

et al. 2019

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With 2129 proteins still classified by the Human Proteome Organisation Human Proteome Project (HPP) as “missing” without compelling evidence of protein existence (PE) in humans, we hypothesized that in-depth proteomic characterization of tissues that are technically challenging to access and extract would yield evidence for tissue-specific missing proteins. Paradoxically, although the skeleton is the most massive tissue system in humans, as one of the poorest characterized by proteomics, bone falls under the HPP umbrella term as a “rare tissue”. Therefore, we aimed to optimize mineralized tissue protein extraction methodology and workflows for proteomic and data analyses of small quantities of healthy young adult human alveolar bone. Osteoid was solubilized by GuHCl extraction, with hydroxyapatite-bound proteins then released by ethylenediaminetetraacetic acid demineralization. A subsequent GuHCl solubilization extraction was followed by solid-phase digestion of the remaining insoluble cross-linked protein using trypsin and then 6 M urea dissolution incorporating LysC digestion. Bone extracts were digested in parallel using trypsin, LysargiNase, AspN, or GluC prior to liquid chromatography–mass spectrometry analysis. Terminal Amine Isotopic Labeling of Substrates was used to purify semitryptic peptides, identifying natural and proteolytic-cleaved neo N-termini of bone proteins. Our strategy enabled complete solubilization of the organic bone matrix leading to extensive categorization of bone proteins in different bone matrix extracts, and hence matrix compartments, for the first time. Moreover, this led to the high confidence identification of pannexin-3, a “missing protein”, found only in the insoluble collagenous matrix and revealed for the first time by trypsin solid-phase digestion. We also found a singleton proteotypic peptide of another missing protein, meiosis inhibitor protein 1. We also identified 17 proteins classified in neXtprot as PE1 based on evidence other than from MS, termed non-MS PE1 proteins, including ≥9-mer proteotypic peptides of four proteins.

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“…The same is also true for sweat (section 7.1) [166,167] and vomit [168]. Forensically relevant solid tissues such as bone [130,134,135,[169][170][171][172][173], teeth (section 7.4) [174][175][176][177][178][179][180][181][182][183][184][185], hair (section 6.6) [9,[186][187][188][189][190][191][192], nail plate [139], and skin cells (section 7.1) [148,193,194], have also been thoroughly processed and analyzed for both forensic as well as clinical purposes. Mineralized and keratinized tissues have also been examined from a paleontological, paleoproteomic perspective that also has forensic relevance for analysis of highly degraded human remains [9,13,130,185,[195][196][197][198][199][200]…”

Section: J O U R N a L P R E -P R O O Fmentioning

confidence: 99%