Unless otherwise stated, all reagents and chemicals were purchased from Sigma-Aldrich.
Blood collection and isolation of PBMCs
All procedures involving human subjects were approved by the Institutional Review Board of the Brody School of Medicine. Venous blood from the brachial region of the upper arm was collected from 30 healthy volunteers, ranging from 18-70 years. Whole blood was collected in 20 sodium-heparinized Cell Preparation Tubes (CPT) (BD Biosciences, Franklin Lakes, NJ) and centrifuged at 1,800 x g for 15 min. Fractions containing peripheral blood mononuclear cells (PBMC) were collected from 4 CPT tubes and used for intact and permeabilized cell experiments while the remaining PBMC fractions were used to isolate mitochondria.
Mononuclear cell isolation from bone marrow aspirates
Bone marrow aspirates were collected from patients undergoing confirmatory diagnosis for a range of hematological malignancies. Patients with confirmed leukemia were enrolled in the study. Type of leukemia ranged from acute myeloid leukemia (AML, N=5), chronic myeloid leukemia (CML, N=2), and granular lymphocytic leukemia. Patient age ranged from 32-78 years (male/female, 3/5). Bone marrow aspirates were compared to PBMC isolated from age-matched participants without a prior history of any hematological malignancy. Peripheral blood and bone marrow aspirates were collected in sodium-heparinized Cell Preparation Tubes (CPT) (BD Biosciences, Franklin Lakes, NJ) and centrifuged at 1,800 x g for 15 min. Mononuclear cells were isolated and then washed in ammonium-chloride-potassium (ACK) lysis buffer to remove red blood cells.
HL-60, KG-1, and MV-4-11 (ATCC, Manassas, VA) human leukemia cells were cultured in IMDM (Thermo Fisher Scientific, Waltham, MA) supplemented with glutamine, 10% FBS, and 1% penicillin/streptomycin and incubated at 37°C in 5% CO2. Upon reaching an average cell density of 1.5x106cell/mL the cells were harvested and used for whole cell and isolated mitochondria experiments. Primary human muscle progenitor cells (human myoblasts, ‘HMB’) were derived from fresh muscle biopsy samples, as described previously61. Cells were cultured on collagen-coated flasks using HMB growth medium (GM: Ham’s F10, supplemented with 20% FBS and 1% penicillin/streptomycin, and supplemented immediately prior to use with 5 ng/ml basic FGF).
Cells were pre-loaded with 200nM Mitotracker Green-FM dye (MTG-FM; Molecular Probes, Eugene, OR) at 37°C for 1hr. Cells were then centrifuged at 300 x g for 7min at ~25°C and resuspended in MTG-FM-free IMDM formulation media (Thermo Fisher) containing 50nM tetramethyl rhodamine methyl ester (TMRM) and 2μM Hoechst 33342. Cells were plated on glass-bottom dishes (MatTek, Ashland, MA) for imaging. Cells were held in place with a thin 1% agarose pad that was applied immediately prior to imaging in order to minimize rapid motion interference during imaging of live non-adherent cells62.
All imaging was performed using an Olympus FV1000 laser scanning confocal microscope (LSCM) with an onstage incubator at 37°C. Acquisition software was Olympus FluoView FSW (V4.2). The objective used was 60X oil immersion (NA=1.35, Olympus Plan Apochromat UPLSAPO60X(F)). Images were 800x800 pixel with 2μs/pixel dwell time, sequential scan mode, resulting in a 4X digital zoom. Hoechst 33342 was excited using the 405nm line of a multiline argon laser; emission was filtered using a 560nm dichroic mirror and 420-460nm barrier filter. MTG-FM was excited using the 488nm line of a multiline argon laser; emission was filtered using a 560nm dichroic mirror and 505-540nm barrier filter. TMRM was excited using a 559nm laser diode; emission was filtered using a 575-675nm barrier filter. Zero detector offset was used for all images and gain at the detectors was kept the same for all imaging. The pinhole aperture diameter was set to 105μm (1 Airy disc).
Images were analyzed using Fiji63. Spatial resolution was measured using sub-resolution fluorescent beads (Thermo Fisher) and curve fitting was performed using the MetroloJ plugin in Fiji. 16-bit images were made into a composite. Circular ROIs were manually selected using the ROI manager plugin. Images were then decomposed into separate 16-bit image stacks leaving the ROI positions intact. A Huang auto-threshold was used for automated selection of signal for all three channels. Following threshold application, each signal was measured using the multi-measure feature. Only whole cells were analyzed (i.e. cells on edges of the FOV were excluded). Slices containing cells above the lowest monolayer were removed from stacks to avoid oversampling. The following calculations were performed to determine the relevant signal volumes.
Signal Volume (μm3) = [A*Z]/N
Where A is the signal-positive area selected using a Huang auto-threshold (μm2), Z is the optical section thickness (axial resolution; μm), and N is the number of steps within each optical section (i.e. axial resolution divided by the step size). The latter operation is necessary to correct for oversampling of the signal volumes.
Respiratory flux in intact and permeabilized cells
Approximately 3 x 106 cells were used for each intact and permeabilized cell experiment. High-resolution respirometry measurements were performed using the Oroboros Oxgraph-2k (O2k; Oroboros Instruments, Innsbruck, Austria) in a 1mL reaction volume at 37°C. At the conclusion of each experiment 1mL of cell suspension was collected from each chamber and centrifuged at 2,000 x g for 10 min at 4°C. Cells were lysed using low-percentage detergent buffer (CelLytic) followed by a freeze-thaw cycle, and protein concentration was determined using a BCA protein assay.
Respiratory flux was measured using previously described methods23. For intact cell measurements, PBMC and leukemia cells were resuspended in Intact Cell Respiratory Media (17.7g/L Iscove’s Modified Dulbecco’s Medium (IMDM), 20mM HEPES, 1% Penicillin/Streptomycin, 10% FBS, pH 7.4). After basal respiration was established, oligomycin (Oligo; 0.02µM) was added followed by FCCP titration (FC; 0.5-5µM), rotenone (Rot; 0.5µM) and antimycin A (Ant; 0.5µM). For permeabilized cell measurements, PBMC and leukemia cells were resuspended in Respiratory Buffer supplemented with creatine (105mM MES potassium salt, 30mM KCl, 8mM NaCl, 1mM EGTA, 10mM KH2PO4, 5mM MgCl2, 0.25% BSA, 5mM creatine monohydrate, pH 7.2). Cells were permeabilized with digitonin (Digi; 0.02mg/mL), and respiratory flux was measured using the creatine kinase (CK) clamp and FCCP titration assays. Within the CK clamp assay, the free energy of ATP hydrolysis (ΔGATP) is calculated using the equilibrium constant for the CK reaction (K’CK) and is based upon the addition of known concentrations of creatine (Cr), phosphocreatine (PCr), and ATP in the presence of excess amounts of CK23. Calculation of ΔGATP at defines PCr concentrations was done using the online resource (https://dmpio.github.io/bioenergetic-calculators/ck_clamp/) previously described23.
For all assays, various combinations of carbon substrates and inhibitors were employed. Substrates and inhibitors utilized are indicated in the figure legends: CK (20U/mL), ATP (5mM), PCr (1mM, 6mM, 15mM, 21mM), pyruvate (5mM), malate (1mM), glutamate (5mM), octanoyl-carnitine (0.2mM), succinate (5mM) cytochrome C (Cyt C, 10µM), oligomycin (Oligo, 0.02µM), FCCP (FC, 0.5-2µM), rotenone (Rot, 0.5µM), antimycin A (Ant A, 0.5µM), carboxyatractyloside (CAT, 1µM), bongkrekic acid (20µM), 17-AAG (sigma, #100068, 1µM), Gamitrinib TPP hexafluorophosphate (MedChemExpress, #HY-102007A, 1µM).
Isolation of mitochondria from PBMCs and leukemia cells
In order to pellet cells, PBMC fractions were washed with PBS and centrifuged at 3,000 x g for 10 min at 4°C and leukemia cells were centrifuged at 300 x g for 10 min followed by a PBS wash. Cell pellets were resuspended in Mitochondrial Isolation Buffer with BSA (100mM KCl, 50mM MOPS, 1mM EGTA, 5mM MgSO4, 0.2% BSA, pH 7.1) and homogenized using a borosilicate glass mortar and Teflon pestle. Homogenates were centrifuged at 800 x g for 10 min at 4°C. The supernatant was collected, and the remaining pellet was resuspended in Mitochondrial Isolation Buffer with BSA, then homogenized and centrifuged again. This process was repeated a total of 3 times. The collected supernatant was centrifuged at 10,000 x g for 10 min at 4°C to pellet the mitochondrial fraction. The fraction was resuspended in Mitochondrial Isolation Buffer without BSA, transferred to a microcentrifuge tube and subjected to a second spin at 10,000 x g. The mitochondrial pellet was resuspended in ~100µL of Mitochondria Isolation Buffer and protein concentration was calculated using the Pierce BCA assay. Respiration assays using isolated mitochondria were similar to that described for permeabilized cells.
Mitochondrial NADH/NAD+ redox in isolated mitochondria
Fluorescent determination of NADH/NAD+ was performed using a QuantaMaster Spectrofluorometer (QM-400, Horiba Scientific, Kyoto, Japan). The NADH/NAD+ was detected at Ex/Em: 350/450. NADH/NAD+ was measured in mitochondria isolated from PBMC and leukemia cell lines using the CK clamp assay. Experiments were performed at 37°C in a 200µL reaction volume. To start, Respiratory Buffer supplemented with creatine (200µL), Cyt C (10µM), mitochondrial lysate (100µg) were added into a glass cuvette. Mitochondria were incubated at 37°C for ~ 5 minutes in the absence of substrate to induce 0% reduction of the NADH pool. Saturating carbon substrates were added (Pyr/Mal/ Oct/Glut/Succ, ‘Multi’), and respiration was stimulated with the CK clamp. Titration of ΔGATP was performed via PCr titration (6, 15, 21mM). Oligomycin (0.02µM) was added to inhibit ATP synthesis and cyanide (CN, 10mM) was added to induce 100% reduction of the matrix NADH pool. The NADH/NAD+ was expressed as a percentage reduction of the CN value (i.e. 100% reduction) based upon the formula % Reduction = (F-F0%)/(F100%-F0%)*100.
Mitochondrial lysis, protein digestion, and peptide labeling for TMT quantitative proteomics
Mitochondrial pellets from leukemia cells and PBMC (approximately 250 µg of protein) were lysed in ice-cold 8 M Urea Lysis Buffer (8 M urea in 50 mM Tris, pH 8.0, 40 mM NaCl, 2 mM CaCl2, 1x cOmplete ULTRA mini EDTA-free protease inhibitor tablet), as described previously36. The samples were frozen on dry ice and thawed for three freeze-thaw cycles and further disrupted by sonication with a probe sonicator in three 5s bursts set at an amplitude of 30 (Q Sonica, Newtown, CT). Samples were centrifuged at 10,000 × g for 10 min at 4 °C to pellet insoluble material. Protein concentration was determined by BCA, and equal amounts of protein (200 μg, adjusted to 2.5 mg/mL with Urea Lysis Buffer) were reduced with 5 mM DTT at 32 °C for 30 min, cooled to room temperature, and then alkylated with 15 mM iodoacetamide for 30 min in the dark. Unreacted iodoacetamide was quenched by the addition of DTT up to 15 mM. Initial digestion was performed with Lys C (Thermo Fisher) 1:100 w-w; 2 µg enzyme per 200 µg protein) for 4 hr at 32 °C. Following dilution to 1.5 M urea with 50 mM Tris (pH 8.0), 30 mM NaCl, 5 mM CaCl2, the samples were digested overnight with trypsin (Promega, Madison, WI) 50:1 w/w, protein:enzyme at 32 °C. Samples were acidified to 0.5% TFA and centrifuged at 10,000 × g for 10 min at 4 °C to pellet insoluble material. Supernatant containing soluble peptides was desalted on a 50 mg tC18 SEP-PAK solid phase extraction column (Waters, Milford, MA) and eluted (500 μL 25% acetonitrile/0.1% TFA and 2 × 500 μL 50% acetonitrile/0.1% TFA). The 1.5 mL eluate was frozen and lyophilized.
TMT labeling was performed as previously described36. The samples from isolated mitochondria were re-suspended in 100 μL of 200 mM triethylammonium bicarbonate (TEAB), mixed with a unique 10-plex Tandem Mass Tag (TMT) reagent (0.8 mg re-suspended in 50 μL100% acetonitrile), and shaken for 4 hr at room temperature (Thermo Fisher). A total of 2 x 10-plex kits were used and one sample was TMT-labeled in both kits to control for quantification differences across multiplex preparations. Following quenching with 0.8 μL 50% hydroxylamine samples were frozen, and lyophilized. Samples were re-suspended in ~1 mL of 0.5% TFA and again subjected to solid phase extraction, but with a 100 mg tC18 SEP-PAK SPE column (Waters). The multiplexed peptide sample was subjected to high pH reversed phase fractionation according to the manufacturer’s instructions (Thermo Fisher). In this protocol, peptides (100 µg) are loaded onto a pH-resistant resin and then desalted with water washing combined with low speed centrifugation. A step-gradient of increasing acetonitrile concentration in a high-pH elution solution is then applied to columns to elute bound peptides into 8 fractions. Following elution, fractions were frozen and lyophilized.
nLC-MS/MS for TMT proteomics
nLC-MS/MS was performed as described previously36. Peptide fractions were suspended in 0.1% formic acid at a concentration of 0.25 µg/µL, following peptide quantification (ThermoFisher). All samples were subjected to nanoLC-MS/MS analysis using an UltiMate 3000 RSLCnano system (Thermo Fisher) coupled to a Q Exactive PlusHybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher) viaa nanoelectrospray ionization source. For each injection of 4 µL (1 µg), the sample was first trapped on an Acclaim PepMapTM100 20 mm × 0.075 mm trapping column (Thermo Fisher) 5 μl/min at 98/2 v/v water/acetonitrile with 0.1% formic acid, after which the analytical separation was performed over a 90-min gradient (flow rate of 300 nanoliters/min) of 3 to 30% acetonitrile using a 2 µm EASY-Spray PepMapTMRSLC C18 75 µm × 250 mm column (Thermo Fisher) with a column temperature of 55 °C. MS1 was performed at 70,000 resolution, with an AGC target of 1 × 106 ions and a maximum IT of 60 ms. MS2 spectra were collected by data-dependent acquisition (DDA) of the top 20 most abundant precursor ions with a charge greater than 1 per MS1 scan, with dynamic exclusion enabled for 30s. Precursor ions were filtered with a 1.0 m/z isolation window and fragmented with a normalized collision energy of 30. MS2 scans were performed at 17,500 resolution, AGC target of 1 × 105 ions, and a maximum IT of 60 ms.
Data analysis for TMT proteomics
Proteome Discoverer 2.2 (PDv2.2) was used for raw data analysis, with default search parameters including oxidation (15.995 Da on M) as a variable modification and carbamidomethyl (57.021 Da on C) and TMT6plex (229.163 Da on peptide N-term and K) as fixed modifications, and 2 missed cleavages (full trypsin specificity). Data were searched against human Mito Carta 2.0 database64. PSMs were filtered to a 1% FDR. PSMs were grouped to unique peptides while maintaining a 1% FDR at the peptide level. Peptides were grouped to proteins using the rules of strict parsimony and proteins were filtered to 1% FDR using the Protein FDR Validator node of PD2.2. MS2 reporter ion intensities for all PSMs having co-isolation interference below 0.5 (50% of the ion current in the isolation window) and an average S/N > 10 for reporter ions were summed together at the peptide and protein level. Imputation was performed via low abundance resampling.
Statistical analysis for TMT proteomic
The protein group tab in the PDv2.2 results was exported as tab delimited.txt. files, and analyzed based on a previously described workflow36. First, M2 reporter (TMT) intensities were summed together for each TMT channel, each channel’s sum was divided by the average of all channels’ sums, resulting in channel-specific loading control normalization factors to correct for any deviation from equal protein input in the 10-plex experiments. Reporter intensities for proteins were divided by the loading control normalization factors for each respective TMT channel. All loading control-normalized reporter intensities were converted to log2 space and the average value from the ten samples per kit was subtracted from each sample specific measurement to normalize the relative measurements to the mean of each kit. Data from each kit were then combined for statistical comparisons. For comparison of PBMC to leukemia cell lines, condition average, standard deviation, p-value (p, two-tailed student’s t-test, assuming equal variance), and adjusted p-value (Padjusted, Benjamini Hochberg FDR correction) were calculated65,66. For protein-level quantification, only Master Proteins—or the most statistically significant protein representing a group of parsimonious proteins containing common peptides identified at 1% FDR—were used for quantitative comparison.
Proteomics data availability and software
All raw data for proteomics experiments is available online using accession number “PXD020715” for Proteome Xchange67 and accession number “JPST000934” for jPOST Repository68.
Statistical Analysis and Software
Statistical analysis was performed using GraphPad Prism 8.4. Among groups, data were analyzed using one-way ANOVA and Tukey’s multiple comparison tests. The assumption of equal variance was assessed using the Brown-Forsythe test. All data are represented as mean ± SEM and analysis were conducted with a significance level set at p<0.05. Details of statistical analysis are included within figure legends. Figures were generated using Biorender and GraphPad Prism 8.4.