Purification of functional mouse skeletal muscle mitochondria using Percoll density gradient centrifugation.

Objective: Our goal was to isolate purified mitochondria from mouse skeletal muscle using a Percoll density gradient and to assess bioenergetic function and purity via Seahorse Extracellular Flux (XF) Analyses and mass spectrometry. Results: Mitochondria isolated from murine quadriceps femoris skeletal muscle using a Percoll density gradient method allowed for minimally contaminated preparations with time from tissue harvest to mitochondrial isolation and quantification in about 3-4 hours. Percoll purification from 100-200 mg fresh tissue yielded ∼200-400 ug protein. Mitochondrial bioenergetics evaluated using the Seahorse XFe96 analyzer, a high-throughput respirometry platform, showed optimum mitochondrial input at 500 ng with respiratory control ratio ranging from 3.9-7.1 using various substrates demonstrating a high degree of functionality. Furthermore, proteomic analysis of Percoll-enriched mitochondria isolated from skeletal muscle using this method showed significant enrichment of mitochondrial proteins indicating high sample purity. This study established a methodology that ensures sufficient high quality mitochondria for downstream analyses such as mitochondrial bioenergetics and proteomics.

Purification of functional mouse skeletal muscle mitochondria using percoll density gradient centrifugation.

OBJECTIVE: Our goal was to isolate purified mitochondria from mouse skeletal muscle using a Percoll density gradient and to assess bioenergetic function and purity via Seahorse Extracellular Flux (XF) Analyses and mass spectrometry. RESULTS: Mitochondria isolated from murine quadriceps femoris skeletal muscle using a Percoll density gradient method allowed for minimally contaminated preparations with time from tissue harvest to mitochondrial isolation and quantification in about 3-4 h. Percoll purification from 100 to 200 mg fresh tissue yielded ~ 200-400 ug protein. Mitochondrial bioenergetics evaluated using the Seahorse XFe96 analyzer, a high-throughput respirometry platform, showed optimum mitochondrial input at 500 ng with respiratory control ratio ranging from 3.9 to 7.1 using various substrates demonstrating a high degree of functionality. Furthermore, proteomic analysis of Percoll-enriched mitochondria isolated from skeletal muscle using this method showed significant enrichment of mitochondrial proteins indicating high sample purity. This study established a methodology that ensures sufficient high quality mitochondria for downstream analyses such as mitochondrial bioenergetics and proteomics.

Exploratory mass spectrometry of cerebrospinal fluid from persons with autopsy-confirmed LATE-NC.

Background: Common neuropathologies associated with dementia include Alzheimer's disease neuropathologic change (ADNC) and limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC). Biofluid proteomics provides a window into the pathobiology of dementia and the information from biofluid tests may help guide clinical management. Methods: Participants were recruited from a longitudinal cohort of older adults at the University of Kentucky AD Research Center. A convenience sample of clinically obtained lumbar puncture cerebrospinal fluid (CSF) samples was analyzed from 29 older adults that had autopsy confirmation of the presence or absence of LATE-NC. Nine of the participants had autopsy-confirmed LATE-NC. Antemortem CSF specimens were analyzed in two separate processes: From one group, aliquots were depleted of highly abundant proteins using affinity spin columns. Tryptic digests of sample proteins were subjected to liquid chromatographic separation and mass spectrometry using an Eksigent Ekspert nanoLC 400 system in line with a Sciex 6600+ mass spectrometer. Protein identification was performed using Protein Pilot (Sciex, ver. 5) software, and relative quantification was performed using the SWATH processing microApp in PeakView and MarkerView software (Sciex), respectively. Following data analyses, additional studies were performed using western blots. Results: A total of 830 proteins were identified in the samples depleted of abundant proteins, and 730 proteins were identified in the non-depleted samples. Whereas some dementia-related proteins were detected (Aß peptide and α-synuclein protein), others were not (TDP-43, TMEM106B, and tau proteins). When the Bonferroni correction was applied to correct for multiple comparisons, only 4 proteins showed differential expression (LATE-NC vs non-LATE-NC) in the nondepleted samples (RBP4, MIF, IGHG3 and ITM2B), whereas none showed statistically different changes in the depleted samples. Post-hoc western blots confirmed that RBP4 expression was higher in the LATE-NC cases at the group level, but there was overlap between the levels of RBP4 in LATE-NC and non-LATE-NC cases. Conclusions: An exploratory assessment of CSF proteomes of autopsy-confirmed LATE-NC and non-LATE-NC cases from a community-based cohort failed to demonstrate a clear-cut proteomic fingerprint that distinguished the two groups. There was intriguing increase in RBP4 protein levels in CSF from LATE-NC cases. This may provide clues about pathogenetic mechanisms in LATE-NC.

Development and characterization of three cell culture systems to investigate the relationship between primary bone marrow adipocytes and myeloma cells.

The unique properties of the bone marrow (BM) allow for migration and proliferation of multiple myeloma (MM) cells while also providing the perfect environment for development of quiescent, drug-resistant MM cell clones. BM adipocytes (BMAds) have recently been identified as important contributors to systemic adipokine levels, bone strength, hematopoiesis, and progression of metastatic and primary BM cancers, such as MM. Recent studies in myeloma suggest that BMAds can be reprogrammed by tumor cells to contribute to myeloma-induced bone disease, and, reciprocally, BMAds support MM cells in vitro. Importantly, most data investigating BMAds have been generated using adipocytes generated by differentiating BM-derived mesenchymal stromal cells (BMSCs) into adipocytes in vitro using adipogenic media, due to the extreme technical challenges associated with isolating and culturing primary adipocytes. However, if studies could be performed with primary adipocytes, then they likely will recapitulate in vivo biology better than BMSC-derived adipocytes, as the differentiation process is artificial and differs from in vivo differentiation, and progenitor cell(s) of the primary BMAd (pBMAds) may not be the same as the BMSCs precursors used for adipogenic differentiation in vitro. Therefore, we developed and refined three methods for culturing pBMAds: two-dimensional (2D) coverslips, 2D transwells, and three-dimensional (3D) silk scaffolds, all of which can be cultured alone or with MM cells to investigate bidirectional tumor-host signaling. To develop an in vitro model with a tissue-like structure to mimic the BM microenvironment, we developed the first 3D, tissue engineered model utilizing pBMAds derived from human BM. We found that pBMAds, which are extremely fragile, can be isolated and stably cultured in 2D for 10 days and in 3D for up to 4 week in vitro. To investigate the relationship between pBMAds and myeloma, MM cells can be added to investigate physical relationships through confocal imaging and soluble signaling molecules via mass spectrometry. In summary, we developed three in vitro cell culture systems to study pBMAds and myeloma cells, which could be adapted to investigate many diseases and biological processes involving the BM, including other bone-homing tumor types.

Proteomic analysis of GLUT4 storage vesicles reveals LRP1 to be an important vesicle component and target of insulin signaling.

Insulin stimulates the translocation of intracellular GLUT4 to the plasma membrane where it functions in adipose and muscle tissue to clear glucose from circulation. The pathway and regulation of GLUT4 trafficking are complicated and incompletely understood and are likely to be contingent upon the various proteins other than GLUT4 that comprise and interact with GLUT4-containing vesicles. Moreover, not all GLUT4 intracellular pools are insulin-responsive as some represent precursor compartments, thus posing a biochemical challenge to the purification and characterization of their content. To address these issues, we immunodepleted precursor GLUT4-rich vesicles and then immunopurified GLUT4 storage vesicle (GSVs) from primary rat adipocytes and subjected them to semi-quantitative and quantitative proteomic analysis. The purified vesicles translocate to the cell surface almost completely in response to insulin, the expected behavior for bona fide GSVs. In total, over 100 proteins were identified, about 50 of which are novel in this experimental context. LRP1 (low density lipoprotein receptor-related protein 1) was identified as a major constituent of GSVs, and we show it interacts with the lumenal domains of GLUT4 and other GSV constituents. Its cytoplasmic tail interacts with the insulin-signaling pathway target, AS160 (Akt substrate of 160 kDa). Depletion of LRP1 from 3T3-L1 adipocytes reduces GLUT4 expression and correspondingly results in decreased insulin-stimulated 2-[(3)H]deoxyglucose uptake. Furthermore, adipose-specific LRP1 knock-out mice also exhibit decreased GLUT4 expression. These findings suggest LRP1 is an important component of GSVs, and its expression is needed for the formation of fully functional GSVs.

Identification of FBXO25-interacting proteins using an integrated proteomics approach.

FBXO25 is one of the 68 human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of s-phase-kinase associated protein 1, really interesting new gene-box 1, Cullin 1, and F-box protein (SCF1) that are involved in targeting proteins for destruction across the ubiquitin proteasome system. We recently reported that the FBXO25 protein accumulates in novel subnuclear structures named FBXO25-associated nuclear domains (FAND). Combining two-step affinity purification followed by MS with a classical two-hybrid screen, we identified 132 novel potential FBXO25 interacting partners. One of the identified proteins, beta-actin, physically interacts through its N-terminus with FBXO25 and is enriched in the FBXO25 nuclear compartments. Inhibitors of actin polymerization promote a significant disruption of FAND, indicating that they are compartments influenced by the organizational state of actin in the nucleus. Furthermore, FBXO25 antibodies interfered with RNA polymerase II transcription in vitro. Our results open new perspectives for the understanding of this novel compartment and its nuclear functions.

A comparison of alternating current and direct current electrospray ionization for mass spectrometry.

A series of studies comparing the performance of alternating current electrospray ionization (AC ESI) mass spectrometry (MS) and direct current electrospray ionization (DC ESI) MS have been conducted, exploring the absolute signal intensity and signal-to-background ratios produced by both methods using caffeine and a model peptide as targets. Because the high-voltage AC signal was more susceptible to generating gas discharges, the operating voltage range of AC ESI was significantly smaller than that for DC ESI, such that the absolute signal intensities produced by DC ESI at peak voltages were one to two orders of magnitude greater than those for AC ESI. Using an electronegative nebulizing gas, sulfur hexafluoride (SF6), instead of nitrogen (N2) increased the operating range of AC ESI by ~50%, but did not appreciably improve signal intensities. While DC ESI generated far greater signal intensities, both ionization methods produced comparable signal-to-background noise, with AC ESI spectra appearing qualitatively cleaner. A quantitative calibration analysis was performed for two analytes, caffeine and the peptide MRFA. AC ESI utilizing SF6 outperforms all other techniques for the detection of MRFA, producing chromatographic limits of detection nearly one order of magnitude lower than that of DC ESI utilizing N2, and one-half that of DC ESI utilizing SF6. However, DC ESI outperforms AC ESI for the analysis of caffeine, indicating that improvements in spectral quality may benefit certain compounds or classes of compounds, on an individual basis.

Catch-and-release reagents for broadscale quantitative proteomics analyses.

The relative quantification of protein expression levels in different cell samples through the utilization of stable isotope dilution has become a standard method in the field of proteomics. We describe here the development of a new reductively cleavable reagent which facilitates the relative quantification of thousands of proteins from only tens of micrograms of starting protein. The ligand features a novel disulfide moiety that links biotin and a thiol-reactive entity. The disulfide is stable to reductive conditions employed during sample labeling but is readily cleaved under mild conditions using tris-(2-carboxyethyl) phosphine (TCEP). This unique chemical property allows for the facile use of immobilized avidin in a manner equivalent to the use of conventional reversible-binding affinity resins. Target peptides are bound to avidin resin, washed rigorously, then cleaved directly from the resin, resulting in simplified sample handling procedures and reduced nonspecific interactions. Here we demonstrate the stability of the linker under two different reducing conditions and show how this "catch-and-release (CAR)" reagent can be used to quantitatively compare protein abundances from two distinct cellular lysates. Starting with only 40 microg protein from each sample, 1840 individual proteins were identified in a single experiment. Using in-house software for automated peak integration, 1620 of these proteins were quantified for differential expression.

Assessing enzyme activities using stable isotope labeling and mass spectrometry.

Activity-based protein profiling has emerged as a valuable technology for labeling, enriching, and assessing protein activities from complex mixtures. This is primarily accomplished via a two-step identification and quantification process. Here we show a highly quantitative and streamlined method, termed catch-and-release activity profiling of enzymes (CAPE), which reduces this procedure to a single step. Furthermore the CAPE approach has the ability to detect small quantitative changes that may have been missed by alternative mass spectrometry-based techniques.

Fluorescent photoaffinity labeling of cytochrome P450 3A4 by lapachenole: identification of modification sites by mass spectrometry.

While photoaffinity ligands (PALs) have been widely used to probe the structures of many receptors and transporters, their effective use in the study of membrane-bound cytochrome P450s is less established. Here, lapachenole has been used as an effective photoaffinity ligand of human P450 3A4, and mass spectrometry data demonstrating the efficient and specific photoaffinity labeling of CYP3A4 by this naturally occurring benzochromene compound is presented. Without photolysis, lapachenole is a substrate of CYP3A4 and can be metabolized to hydroxylated products by this enzyme. A high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) procedure was developed to analyze small amounts of intact purified CYP3A4, and analysis of the labeled protein showed the presence of one molecule of lapachenole bound per monomer of protein. Photolabeled CYP3A4 peptide adducts were further characterized by mass spectrometric analysis after proteolytic digestion and isolation of fluorescent photolabeled peptides. Two peptide adducts accounting for >95% of the labeled peptides were isolated by HPLC, and both peptides, ECYSVFTNR (positions 97-105) and VLQNFSFKPCK (positions 459-469), were identified by nano-LC/ESI quadrupole time-of-flight (QTOF) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The sites of modification were further localized to positions Cys-98 and Cys-468 for each peptide by nano-LC/ESI QTOF tandem mass spectrometry (MS/MS). The results provided the first direct evidence for interaction between the PAL and the putative B-B' loop region, which may serve as a substrate access channel or as a part of the CYP3A4 active site. In conclusion, benzochromene analogues are effective PALs, which may be used in the study of other cytochrome P450 structures.

Photochromic agents as tools for protein structure study: lapachenole is a photoaffinity ligand of cytochrome P450 3A4.

Cytochrome P450 3A4 is a drug-metabolizing enzyme of extraordinarily broad substrate specificity. This quality imparts upon the enzyme special importance in understanding its determinants of activity and substrate recognition. Limited successes in P450 3A4 active-site structure studies have been achieved by use of mechanism-based inactivators and photoaffinity ligands. We report here the potential of photochromic agents, compounds with the ability to undergo light-induced, reversible reactions, to be used as effective photoaffinity ligands. Four such compounds of the chromene family were shown by ultraviolet and visible spectroscopy to undergo photoinduced rearrangements to highly conjugated and reactive products in buffered aqueous solution. While some of these intermediates were very long-lived (>12 h, photoactivated lapachenole), others existed for milliseconds in their opened forms (precocene I and 2,2-dimethyl-5,6-benzo-2H-chromene) and were observed by laser flash photolysis. Each of the tricyclic structures studied rapidly underwent Michael addition reactions with the test nucleophile glutathione upon irradiation to form single conjugated products. The smaller precocene I reacted more extensively to form multiple products. These attributes of the chromenes inspired testing of their potential to label cytochrome P450 3A4 in a light-dependent fashion. Access to the protein active site by lapachenole was demonstrated with the molecule's ability to competitively inhibit P450 3A4-mediated oxidative metabolism of midazolam with an IC(50) value of 71 microM. This inhibition became irreversible upon irradiation of the enzyme-ligand complex with ultraviolet light. These results clearly demonstrate that chromenes are effective photoaffinity reagents for the cytochrome P450 superfamily of enzymes and probably other proteins as well.