An 8-plex version of an isobaric reagent for the quantitation of proteins using shotgun methods is presented. The 8-plex version of the reagent relies on amine-labeling chemistry of peptides similar to 4-plex reagents. MS/MS reporter ions at 113, 114, 115, 116, 117, 118, 119, and 121 m/z are used to quantify protein expression. This technology which was first applied to a test mixture consisting of 8 proteins and resulted in accurate quantitation, has the potential to increase throughput of analysis for quantitative shotgun proteomics experiments when compared to 2-plex and 4-plex methods. The technology was subsequently applied to a longitudinal study of cerebrospinal fluid proteins from subjects undergoing intravenous immunoglobulin treatment for Alzheimer’s disease. Results from this study identify a number of protein expression changes that occur in cerebrospinal fluid after 3 and 6 months of treatment compared to a baseline and compared to a drug washout period. A visualization tool was developed for this dataset and is presented. The tool can aid in the identification of key peptides and measurements. One conclusion aided by the visualization tool is that there are differences in considering peptide-based observations versus protein-based observations from quantitative shotgun proteomics studies.

Efficient extraction and accurate quantification of nucleolar macromolecules are critical for in vitro analysis, especially for studying RNA, DNA, and protein dynamics under identical conditions. There is presently no single method that efficiently and simultaneously isolates these three macromolecular constituents from purified nucleoli. We have developed an optimized method, which without evident loss, extracts, and solubilizes protein recovered from a single sample following TRIzol isolation of RNA and DNA. The solubilized protein can be accurately quantified by protein bicinchoninic acid assay and assessed by polyacrylamide gel electrophoresis. We have successfully applied this approach to extract and quantify all three nucleolar components, and to study nucleolar protein responses after actinomycin D treatment.

Almost all primary transcripts in higher eukaryotes undergo several splicing events and alternative splicing is a major factor in generating proteomic diversity. Thus, the spliceosome, the ribonucleoprotein assembly that performs splicing, is a highly critical cellular machine and as expected, a very complex one. Indeed, the spliceosome is one of the largest, if not the largest, molecular machine in the cell with over 150 different components in human. A large fraction of the spliceosomal proteome is organized into ribonucleoprotein particles (snRNPs) by associating with one of the small nuclear RNAs (snRNAs), and the function of many spliceosomal proteins revolve around their association or interaction with the spliceosomal RNAs or the substrate pre-messenger RNAs. In addition to the complex web of protein-RNA interactions, an equally complex network of protein-protein interactions exists in the spliceosome which includes a number of large, conserved proteins with critical functions in the spliceosomal catalytic core. These include the largest conserved nuclear protein, Prp8, which plays a critical role in spliceosomal function in a hitherto unknown manner. Taken together, the large spliceosomal proteome and its dynamic nature has made it a highly challenging system to study, and at the same time, provides an exciting example of the evolution of a proteome around a backbone of primordial RNAs likely dating from the RNA World.

Human serum glycomics is a promising method for finding cancer biomarkers but often lacks the tools for streamlined data analysis. The Glycolyzer software incorporates a suite of analytic tools capable of identifying informative glycan peaks out of raw mass spectrometry data. As a demonstration of its utility, the program was used to identify putative biomarkers for epithelial ovarian cancer from a human serum sample set. A randomized, blocked and blinded experimental design was used on a discovery set consisting of 46 cases and 48 controls. Retrosynthetic glycan libraries were used for data analysis and several significant candidate glycan biomarkers were discovered via hypothesis testing. The significant glycans were attributed to a glycan family based on glycan composition relationships and incorporated into a linear classifier motif test. The motif test was then applied to the discovery set to evaluate the disease state discrimination performance. The test provided strongly predictive results based on receiver operator characteristic curve analysis. The area under the receiver operator characteristic curve was 0.93. Using the Glycolyzer software, we were able to identify a set of glycan biomarkers that highly discriminate between cases and controls, and are ready to be formally validated in subsequent studies.

Receptor tyrosine kinases (RTKs) are proteins that upon ligand stimulation undergo dimerization and autophosphorylation. Eph receptors (EphRs) are RTKs that are found in different cell types, from both tissues that are developing and from mature tissues and play important roles in the development of the central nervous system and peripheral nervous system. EphRs also play roles in synapse formation, neural crest formation, angiogenesis and in remodeling the vascular system. Interaction of EphRs with their ephrin ligands leads to activation of signal transduction pathways and to formation of many transient protein-protein interactions that ultimately leads to cytoskeletal remodeling. However, the sequence of events at the molecular level is not well-understood.

We used Blue Native PAGE (BN-PAGE) and mass spectrometry (MS) to analyze the transient protein-protein interactions that resulted from stimulation of EphB2 receptors by their ephrinB1-Fc ligands. We analyzed the phosphotyrosine-containing protein complexes immunoprecipitated (pY-IPs) from the cell lysates of both unstimulated (−) and ephrinB1-Fc-stimulated (+) NG108 cells. Our experiments allowed us to identify many signaling proteins, either known to be part of EphB2 signaling or new for this pathway, which are involved in transient protein-protein interactions upon ephrinB1-Fc stimulation. These data led us to investigate the roles in EphB2 signaling of proteins such as FAK, WAVEs, and Nischarin.

We describe a novel two-step LC/MSn strategy to effectively and confidently identify numerous crosslinked peptides from complex mixtures. This method incorporates the use of our gas-phase cleavable crosslinking reagent, disuccinimidyl-succinamyl-aspartyl-proline (SuDP), and a new data processing algorithm CXLinkS (Cleavable Crosslink Selection), which enables unequivocal crosslink peptide selection and identification on the basis of mass measurement accuracy, high resolving power, and the unique fragmentation pattern of each crosslinked peptide. We demonstrate our approach with well-characterized monomeric and multimeric protein systems with and without database searching restrictions where inter-peptide crosslink identification is increased 8-fold over our previously published data-dependent LC/MS3 method and discuss its applicability to other CID-cleavable crosslinkers and more complex protein systems.

Proteases play prominent roles in many physiological processes and the pathogenesis of various diseases, which makes them interesting drug targets. To fully understand the functional role of proteases in these processes, it is necessary to characterize the target specificity of the enzymes, identify endogenous substrates and cleavage products as well as protease activators and inhibitors. The complexity of these proteolytic networks presents a considerable analytic challenge. To comprehensively characterize these systems, quantitative methods that capture the spatial and temporal distributions of the network members are needed. Recently, activity-based workflows have come to the forefront to tackle the dynamic aspects of proteolytic processing networks in vitro, ex vivo and in vivo. In this review, we will discuss how mass spectrometry-based approaches can be used to gain new insights into protease biology by determining substrate specificities, profiling the activity-states of proteases, monitoring proteolysis in vivo, measuring reaction kinetics and defining in vitro and in vivo proteolytic events. In addition, examples of future aspects of protease research that go beyond mass spectrometry-based applications are given.

The proteins secreted by various cells (the secretomes) are a potential rich source of biomarkers since they reflect various states of the cells at real time and at given conditions. To have accessible, sufficient and reliable protein markers is desirable since they mark various stages of disease development and their presence/absence can be used for diagnosis, prognosis, risk stratification and therapeutic monitoring. As direct analysis of blood/plasma, a common and noninvasive patient screening method, can be difficult for candidate protein biomarker identification, the alternative/complementary approaches are required, one of them is the analysis of secretomes in cell conditioned media in vitro. Since the proteins secreted by cells as a response to various stimuli are most likely secreted into blood/plasma, the identification and preselection of candidate protein biomarkers from cell secretomes with subsequent validation of their presence at higher levels in serum/plasma is a promising approach. In this review, we discuss the proteins secreted by three progenitor cell types (smooth muscle, endothelial and cardiac progenitor cells) and two adult cell types (neonatal rat ventrical myocytes and smooth muscle cells) which can be relevant to cardiovascular research and which have been recently published in the literature. We found, at least for secretome studies included in this review, that secretomes of progenitor and adult cells overlap by 48% but the secretomes are very distinct among progenitor cell themselves as well as between adult cells. In addition, we compared secreted proteins to protein identifications listed in the Human Plasma PeptideAtlas and in two reports with cardiovascular-related proteins and we performed the extensive literature search to find if any of these secreted proteins were identified in a biomarker study. As expected, many proteins have been identified as biomarkers in cancer but 18 proteins (out of 62) have been tested as biomarkers in cardiovascular diseases as well.

While the use of detergents is necessary for a variety of protein isolation preparation protocols, they are not compatible with mass spectral analysis due to ion suppression and adduct formation. This manuscript describes optimization of detergent removal, using commercially available SDS depletion spin columns containing an affinity resin, providing for both increased protein recovery and thorough SDS removal. Ion mobility spectrometry coupled with mass spectrometry (IMS-MS) allowed for a concurrent analysis of both analyte and detergent. In the case of both proteins and peptides, higher detergent concentrations than previously reported provided an increase of sample recovery; however there was a limit as SDS was detected by IMS-MS at higher levels of SDS indicating incomplete detergent depletion. The results also suggest optimal conditions for SDS removal are dependent on the sample concentration. Overall, this study provides a useful guide for proteomic studies where SDS is required for efficient sample preparation.

The strength of the streptavidin/biotin interaction poses challenges for the recovery of biotinylated molecules from streptavidin resins. As an alternative to high temperature elution in urea containing buffers, we show mono-biotinylated proteins can be released with relatively gentle heating in the presence of biotin and 2% SDS/Rapigest, avoiding protein carbamylation and minimizing streptavidin dissociation. We demonstrate the utility of this mild elution strategy in two studies of the human androgen receptor (AR). In the first, in which formaldehyde crosslinked complexes are analyzed in yeast, a mass spectrometry-based comparison of the AR complex using SILAC reveals an association between the androgen activated AR and the Hsp90 chaperonin, while Hsp70 chaperonins associate specifically with the unliganded complex. In the second study, the endogenous AR is quantified in the LNCaP cell line by absolute SILAC and MRM-MS showing approximately 127,000 AR copies per cell, substantially more than previously measured using radioligand binding.

This paper considers the problem of optimal false discovery rate control when the test statistics are dependent. An optimal joint oracle procedure, which minimizes the false non-discovery rate subject to a constraint on the false discovery rate is developed. A data-driven marginal plug-in procedure is then proposed to approximate the optimal joint procedure for multivariate normal data. It is shown that the marginal procedure is asymptotically optimal for multivariate normal data with a short-range dependent covariance structure. Numerical results show that the marginal procedure controls false discovery rate and leads to a smaller false non-discovery rate than several commonly used p-value based false discovery rate controlling methods. The procedure is illustrated by an application to a genome-wide association study of neuroblastoma and it identifies a few more genetic variants that are potentially associated with neuroblastoma than several p-value-based false discovery rate controlling procedures.

Sites of ubiquitin modification have been identified by mass spectrometry based on the increase in molecular mass of a tryptic peptide carrying two additional glycine residues from the ubiquitin moiety. However, such peptides with GG shifts have been difficult to discover. We identify 870 unique sites of ubiquitin attachment on 438 different proteins of the yeast Saccharomyces cerevisiae.

Amidation is a post-translational modification found at the C-terminus of ~50% of all neuropeptide hormones. Cleavage of the Cα-N bond of a C-terminal glycine yields the α-amidated peptide in a reaction catalyzed by peptidylglycine α-amidating monooxygenase (PAM). The mass of an α-amidated peptide decreases by 58 Da relative to its precursor. The amino acid sequences of an α-amidated peptide and its precursor differ only by the C-terminal glycine meaning that the peptides exhibit similar RP-HPLC properties and tandem mass spectral (MS/MS) fragmentation patterns. Growth of cultured cells in the presence of a PAM inhibitor ensured the coexistence of α-amidated peptides and their precursors. A strategy was developed for precursor and α-amidated peptide pairing (PAPP): LC-MS/MS data of peptide extracts were scanned for peptide pairs that differed by 58 Da in mass, but had similar RP-HPLC retention times. The resulting peptide pairs were validated by checking for similar fragmentation patterns in their MS/MS data prior to identification by database searching or manual interpretation. This approach significantly reduced the number of spectra requiring interpretation, decreasing the computing time required for database searching and enabling manual interpretation of unidentified spectra. Reported here are the α-amidated peptides identified from AtT-20 cells using the PAPP method.

Exosomes are membrane vesicles that are secreted by cells upon fusion of multivesicular bodies with the plasma membrane. Exosomal proteomics has emerged as a powerful approach to understand the molecular composition of exosomes and has potential to accelerate biomarker discovery. Different proteomic analysis methods have been previously employed to establish several exosome protein databases. In this study, TFE solution phase digestion was compared with in-gel digestion and found to yield similar results. Proteomic analysis of urinary exosomes was performed by multidimensional protein identification technology (MudPIT) after TFE digestion. 3280 proteins were identified from nine human urine samples with 31% overlap among nine samples. Gene ontology (GO) analysis, coupled with detection of all of the members of ESCRT machinery complex, supports the multivesicular origin of these particles. These results significantly expand the existing database of urinary exosome proteins. Our results also indicate that more than 1000 proteins can be detected from exosomes prepared from as little as 25 mL of urine. This study provides the largest set of proteins present in human urinary exosome proteomes, provides a valuable reference for future studies, and provides methods that can be applied to exosomal proteomic analysis from other tissue sources.

The transgenic adenocarcinoma of mouse prostate (TRAMP) is the most widely used transgenic model for prostate cancer chemoprevention studies. Although two lobe-specific lineages of carcinogenesis have been described, the molecular mechanisms are still poorly defined. Here, we concurrently profiled the proteome of dorsal-lateral (DLP) and ventral (VP) prostate lobes of both TRAMP and littermate wild type C57BL/6 mice of 18 weeks by 2D-LC-MALDI-TOF/TOF with iTRAQ labeling. A total of 483 and 748 proteins were identified at critical false discovery rates of 1% and 5%. In wild-type mice, 84 proteins were found to have different expression levels between DLP and VP. In TRAMP mice, 118 proteins significantly changed in DLP and/or VP during TRAMP carcinogenesis. Among them, 55 and 36 proteins were uniquely changed in DLP or VP lobe, respectively and 27 proteins in both DLP and LP lobe. Ingenuity Pathway Analysis (IPA) was able to segregate proteins changed in two lobes into different pathway networks. In addition to serving as reference for prostate proteomic profiles, our data suggest that different sets of proteins are involved in the carcinogenesis in DLP vs. in VP in the TRAMP model.

The use of isobaric tags such as iTRAQ allows the relative and absolute quantification of hundreds of proteins in a single experiment for up to eight different samples. More classical techniques such as 2-DE can offer a complimentary approach for the analysis of complex protein samples. In this study, the proteomes of secreted and cytosolic proteins of genetically closely related strains of Mycobacterium tuberculosis were analyzed. Analysis of 2-D gels afforded 28 spots with variations in protein abundance between strains. These were identified by tandem MS/MS. Meanwhile, a rigorous statistical analysis of iTRAQ data allowed the identification and quantification of 101 and 137 proteins in the secreted and cytosolic fractions respectively. Interestingly, several differences in protein levels were observed between the closely related strains BE, C28 and H6. Seven proteins related to cell wall and cell processes were more abundant in BE, while enzymes related to metabolic pathways (GltA2, SucC, Gnd1, Eno) presented lower levels in the BE strain. Proteins involved in iron and sulfur acquisition (BfrB, ViuB, TB15.3 and SseC2) were more abundant in C28 and H6. In general, iTRAQ afforded rapid identification of fine differences between protein levels such as those presented between closely related strains. This provides a platform from which the relevance of these differences can be assessed further using complimentary proteomic and biological modeling methods.

The current best serum marker for pancreatic cancer, CA 19-9, detects a carbohydrate antigen on multiple protein carriers. Better knowledge of the protein carriers of the CA 19-9 antigen in various disease states may lead to improved diagnostic tests. To identify proteins that carry the CA 19-9 antigen, we immunoprecipitated the CA 19-9 antigen from pooled sera and identified the associated proteins using mass spectrometry. Among the high-confidence identifications, we confirmed the presence of the CA 19-9 antigen on Apolipoprotein B-100 by antibody arrays and Western blot and on kininogen, ARVCF, and Apolipoprotein E by antibody arrays. We characterized the frequency and levels of the CA 19-9 antigen on the four proteins across various patient groups (pancreatic cancer, pancreatitis, and healthy controls) using antibody arrays. 10–25% of the subjects showed elevations of the antigen on each protein, but the elevations were not associated with disease state or total CA 19-9 levels. These results contribute to our knowledge of the carrier proteins of an important functional glycan and the rate at which the glycan is displayed. This work also demonstrates a strategy for using the complementary methods of mass spectrometry and antibody microarrays to identify protein carriers of glycans and assess the diagnostic value of measuring glycans on individual proteins.

Proteomics analyses were performed on the brains of wild-type (WT) controls and an Alzheimer’s disease (AD) mouse model, APP/PS-1 human double mutant knock in mice. Mice were given either drinking water or water supplemented with N-acetylcysteine (NAC) (2mg/kg body weight) for a period of five months. The time periods of treatment correspond to ages prior to Aβ deposition (i.e., 4–9 months), resembling human mild cognitive impairment (MCI), and after Aβ deposition (i.e., 7–12 months), more closely resembling advancing stages of AD. Substantial differences exist between the proteomes of WT and APP/PS-1 mice at 9 or 12 months, indicating that Aβ deposition and oxidative stress lead to downstream changes in protein expression. Altered proteins are involved in energy-related pathways, excitotoxicity, cell cycle signaling, synaptic abnormalities, and cellular defense and structure. Overall, the proteomic results support the notion that NAC may be beneficial for increasing cellular stress responses in WT mice and for influencing the levels of energy- and mitochondrial related proteins in APP/PS-1 mice.

Pancreatic cancer is a deadly disease characterized by poor prognosis and patient survival. Green tea polyphenols have been shown to exhibit multiple antitumor activities in various cancers, but studies on the pancreatic cancer are very limited. To identify the cellular targets of green tea action, we exposed a green tea extract (GTE) to human pancreatic ductal adenocarcinoma HPAF-II cells and performed two-dimensional gel electrophoresis of the cell lysates. We identified 32 proteins with significantly altered expression levels. These proteins are involved in drug resistance, gene regulation, motility, detoxification and metabolism of cancer cells. In particular, we found GTE inhibited molecular chaperones heat-shock protein 90 (Hsp90), its mitochondrial localized homologue Hsp75 (tumor necrosis factor receptor-associated protein 1, or Trap1) and heat-shock protein 27 (Hsp27) concomitantly. Western blot analysis confirmed the inhibition of Hsp90, Hsp75 and Hsp27 by GTE, but increased phosphorylation of Ser78 of Hsp27. Furthermore, we showed that GTE inhibited Akt activation and the levels of mutant p53 protein, and induced apoptosis and growth suppression of the cells. Our study has identified multiple new molecular targets of GTE and provided further evidence on the anticancer activity of green tea in pancreatic cancer.

The array of biomolecules generated by a functioning ecosystem represents both a potential resource for sustainable harvest and a potential indicator of ecosystem health and function. The cupped leaves of the carnivorous pitcher plant, Sarracenia purpurea, harbor a dynamic food web of aquatic invertebrates in a fully functional miniature ecosystem. The energetic base of this food web consists of insect prey, which is shredded by aquatic invertebrates and decomposed by microbes. Biomolecules and metabolites produced by this food web are actively exchanged with the photosynthesizing plant. In this report, we provide the first proteomic characterization of the sacrophagid fly (Fletcherimyia fletcheri), the pitcher plant mosquito (Wyeomyia smithii), and the pitcher-plant midge (Metriocnemus knabi). These three arthropods act as predators, filter feeders, and shredders at distinct trophic levels within the S. purpurea food web. More than 50 proteins from each species were identified, 10 of which were predominantly or uniquely found in one species. Furthermore, 19 peptides unique to one of the three species were identified using an assembled database of 100 metazoan myosin heavy chain orthologs. These molecular signatures may be useful in species monitoring within heterogeneous ecosystem biomass and may also serve as indicators of ecosystem state.

Enterococcus faecalis is a gram-positive bacterium that is part of the indigenous microbiotica of humans and animals as well as an opportunistic pathogen. In this study we have fractionated the membrane proteome of E. faecalis and identified many of its constituents by mass spectrometry. We present BN-/SDS-PAGE reference maps that contain 102 proteins. These proteins are important for cellular homeostasis, virulence, and antibiotic intervention. Intriguingly, many proteins with no known function were also identified, indicating that there are substantial gaps in knowledge of this organism’s biology. On a more limited scale we also provide insight into the composition of membrane protein complexes. This study is a first step toward elucidating the membrane proteome of E. faecaliswhich is critical for a better understanding of how this bacterium interacts with a host and with the extracellular milieu.

Notch Signaling has been demonstrated to have a central role in Glioblastoma (GBM) Cancer Stem Cells (CSCs) and we have demonstrated recently that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes GBM CSCs and prevents tumor propagation both in vitro and in vivo. In order to understand the proteome alterations involved in this transformation, a dose-dependent quantitative mass spectrometry (MS) based proteomic study has been performed based on global proteome profiling and a target verification phase where both Immunoassay and a Multiple Reaction Monitoring (MRM) assay are employed. The selection of putative protein candidates for confirmation poses a challenge due to the large number of identifications from the discovery phase. A multilevel filtering strategy together with literature mining is adopted to transmit the most confident candidates along the pipeline. Our results indicate that treating GBM CSCs with GSI induces a phenotype transformation towards non-tumorigenic cells with decreased proliferation and increased differentiation, as well as elevated apoptosis. Suppressed glucose metabolism and attenuated NFR2-mediated oxidative stress response are also suggested from our data, possibly due to their crosstalk with Notch Signaling. Overall, this quantitative proteomic based dose-dependent work complements our current understanding of the altered signaling events occurring upon the treatment of GSI in GBM CSCs.

High-resolution top-down mass spectrometry was used to characterize eleven integral and five peripheral subunits of the 750 kDa Photosystem II (PSII) complex from the eukaryotic red alga, Galdieria sulphuraria. The primary separation used liquid chromatography mass spectrometry with concomitant fraction collection (LC-MS+) yielding around 40 intact mass tags (IMTs) at 100 ppm mass accuracy on a low-resolution electrospray-ionization mass spectrometer, whose retention and mass were used to guide subsequent high-resolution top-down nano-electrospray Fourier-transform ion-cyclotron resonance mass spectrometry experiments (FT-MS). Both collisionally activated and electron capture dissociation (CAD, ECD) were used to confirm the presence of eleven small subunits to mass accuracy within 5 ppm; PsbE, PsbF, PsbH, PsbI, PsbJ, PsbK, PsbL, PsbM, PsbT, PsbX and PsbZ. All subunits showed covalent modifications that fall into three classes including retention of initiating formyl-methionine, removal of methionine at the N-terminus with or without acetylation, and removal of a longer N-terminal peptide. Peripheral subunits identified by top-down analysis included oxygen evolving complex (OEC) subunits PsbO, PsbU, PsbV, as well as Psb28 (PsbW) and Psb27 (‘PsbZ-like’). Top-down high-resolution mass spectrometry provides the necessary precision, typically less than 5 ppm, for identification and characterization of polypeptide composition of these important membrane protein complexes.

In a typical shotgun proteomics experiments, a significant number of high quality MS/MS spectra remain “unassigned”. The main focus of this work is to improve our understanding of various sources of unassigned high quality spectra. To achieve this, we designed an iterative computational approach for more efficient interrogation of MS/MS data. The method involves multiple stages of database searching with different search parameters, spectral library searching, blind searching for modified peptides, and genomic database searching. The method is applied to a large publicly available shotgun proteomic dataset.

Since little is known regarding osteocytes, cells embedded within the mineralized bone matrix, a proteomics approach was used to discover proteins more highly expressed in osteocytes than in osteoblasts to determine osteocyte specific function. Two proteomic profiles obtained by two different proteomic approaches using total cell lysates from the osteocyte cell line MLO-Y4 and the osteoblast cell line MC3T3 revealed unique differences. Three protein clusters, one related to glycolysis, (Phosphoglycerate kinase 1, fructose-bisphosphate aldolase A, hypoxia up-regulated 1 [ORP150], triosephosphate isomerase), one to protein folding (Mitochondrial Stress-70 protein, ORP150, Endoplasmin), and one to actin cytoskeleton regulation (Macrophage-capping protein [CapG], destrin, forms of lamin A and vimentin) were identified. Higher protein expression of ORP-150, Cap G, and destrin in MLO-Y4 cells compared to MC3T3 cells was validated by gene expression, Western blotting, and in vivo expression. These proteins were shown to be selective in osteocytes in vivo using immuno-staining of mouse ulnae. Destrin was most highly expressed in embedding osteoid osteocytes, GapG in embedded osteocytes, and ORP150 in deeply embedded osteocytes. In summary, the proteomic approach has yielded important information regarding molecular mechanisms used by osteocytes for embedding in matrix, the formation of dendritic processes, and protection within a hypoxic environment.