Allgrove-patient derived and control skin fibroblasts and muscle biopsy specimen
The Allgrove-patient was identified early in the second decade of life when declining school performance together with mild dysmorphic features lead to suggestion of a neurological disorder. He presented with the well-described signs of type II achalasia and the combination of predominately distal muscle weakness and atrophy with brisk deep tendon reflexes. Clinical severity and manifesting age define this as a case of intermediate severity, although there is no established grading system.
The muscle biopsy derived from this genetically confirmed Allgrove patient (c.762delC mutation in the nucleoporin gene AAAS; (10)) was collected for diagnostic purpose including histological and electron microscopic investigations. In the same procedure, a skin biopsy was collected and used to set-up a fibroblast culture now available for further functional studies focusing on the cellular impact of loss of functional ALADIN. Primary human fibroblasts from the Allgrove-patient and controls (n = 3; obtained from the MRC Centre Neuromuscular Biobank Newcastle (40)) were cultured in minimum essential media supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 2 mM L‐glutamine, 1x non‐essential amino acids, 1x minimum essential medium vitamins, 1 mM sodium pyruvate, 50 μg/mL uridine (ThermoFisher Scientific), at 37 °C, in a humidified 5% CO2 atmosphere. Cells were grown to a confluency of 70% prior collection for proteomic studies: cells were collected in a 1.5 ml tube, washed twice with ice-cold PBS, snap-frozen in liquid nitrogen and stored at -80 °C until protein extraction for subsequent proteomic studies (see below).
Sample preparation of fibroblasts for spectral library generation
Snap-frozen fibroblasts were lysed in a buffer containing 1% SDS, 50 mM Tris, 150 mM NaCl, pH 7.8 and cOmplete™ ULTRA protease inhibitor using the Bioruptor® (Diagenode) for 10 minutes (30 seconds on, 30 seconds off, 10 cycles) at 4 °C. Afterwards, 20 µl of each sample was taken and diluted 1:4 with 10 mM ammoniumbicarbonate buffer, pH 7.8 (ABC) to conduct a BCA-based determination of protein concentration according to the manufacturer’s instructions (Pierce BCA protein assay kit). Reduction and carbamidomethylation of the remaining samples were carried out using 10 mM Tris-(2-carboxyethyl)-phosphin (TCEP) for 30 min at 37 C followed by application of 15 mM Iodacetamide (IAA) for further 30 min at room temperature (RT).
Samples were further processed using the S-Trap™ (Protifi) sample preparation procedure: after acidifying the samples by adding 12% aqueous phosphoric acid, they were diluted with S-Trap binding buffer (90% methanol (MeOH), triethylammonium bicarbonate (TEAB) 100 mM, pH 7.1). Protein-loading to S-Trap columns including centrifugation steps was performed according to the manufacturer’s instructions. Filter-based tryptic digestion was carried out for 2 h at 47 °C using a trypsin to protein ratio of 1:20. Afterwards, peptides were eluted by applying multiple eluting steps starting with 10 mM ABC followed by elution with 0.1% formic acid (FA) and last with 80% acetonitrile (ACN). Drying of the eluted peptides was performed in a vacuum concentrator followed by dissolving of peptides in 0.1% TFA for subsequent LC-MS/MS analysis or 10 mM ammonium acetate with 0.4 mM FA (pH 8.0) for subsequent pH8 reversed phase fractionation.
All proteolytic digests were analyzed using monolithic column separation system (PepSwift monolithic PS-DVB PL-CAP200-PM, Dionex) on an inert Ultimate 3000 HPLC (Dionex) by direct injection of 1 μg sample to proof effectiveness of tryptic digestion. A binary gradient (solvent A: 0.1% TFA, solvent B: 0.08% TFA, 84% ACN) ranging from 5-12% B in 5 min and then from 12-50% B in 15 min at a flow rate of 2.2 μl/min and at 60 °C, was applied. UV traces were acquired at 214 nm (41).
PH8-based sample fractionation
Each of the digested and desalted samples selected for the following generation of a spectral library was first dried using a vacuum concentrator. The peptides were then dissolved in a buffer containing 10 mM ammonium acetate and 0.4 mM formiate (pH 8.0) (concentration 50μg/μl) and separated on a C18 RP chromatography column (loading amount 50 μg). Doing so, peptides were loaded onto the column with solvent A (10 mM ammonium acetate, 0.4 mM formiate, pH 8.0) at a flow rate of 12.5 μl/min. Separation and fractionation were performed with the following gradient with solvent B (84% acetonitrile in 10 mM ammonium acetate, 0.4 mM formiate, pH 8.0): 3-10% in 10 min, 10-25% for 35 min, 25-40% for 20 min, 40-95% for 10 min, 95% for 5 min and 20 min equilibration at 3%. The individual fractions were collected in an interval of 60 s, with each sample divided into 15 fractions. Collection was performed in the time interval of 10 to 75 min of the gradient. The fractions were collected in combined form, so that after reaching the last fraction, the sample was collected again for one minute in fraction 1. After fractionation, the individual samples were dried in a vacuum concentrator and dissolved in 0.1% TFA prior subsequent nano LC-MS/MS analysis (1 μg/μl).
Spectral library generation
Given that setting up a spectral library is a prerequisite to perform a data independent LC-MS/MS-based sample analysis, all fractions derived from the pH8 fractionation mentioned above were analyzed by nano LC-MS/MS using 1 µg respectively: samples were loaded on an Ultimate 3000 Rapid Separation Liquid chromatography (RSLC) nano system with a ProFlow flow control device coupled to a Fusion Lumos Tribrid mass spectrometer (both from Thermo Scientific). After initial loading, peptides were concentrated on a trapping column (Acclaim C18 PepMap100, 100 µm, 2 cm) using 0.1% TFA at a flowrate of 10 µl/min. Following sample separation was accomplished on a reversed phase column (Acclaim C18 PepMap100, 75 µm 50 cm) using a binary gradient: 3% solvent B (84% ACN with 0.1% TFA) for 10 min, a linear increase of solvent B to 35% for 120 min, a linear increase of solvent B to 95% for 10 min followed by a linear decrease of solvent B to 3% for 5 min. MS survey scans were acquired on the Fusion Lumos using settings as follows: mass spectrometer was operated in data dependent acquisition mode (DDA) with full MS scans from 300 to 1500 m/z at a resolution of 120,000 (Orbitrap) using the polysiloxane ion at 445.12002 m/z as lock mass. The automatic gain control (AGC) was set to 2E5 and the maximum injection time to 50 milliseconds. The most intense ions above a threshold ion count of 5E3 were selected for fragmentation at a normalized collision energy (nCE) of 30% (HCD) in each cycle of the acquisition analysis, following each survey scan. The dynamic exclusion time was set to 15 seconds. The number of selected precursor ions for fragmentation was determined by the “rapid” acquisition algorithm. Fragment ions were acquired in the linear ion trap with an AGC of 1E4 and a maximum injection time of 35 milliseconds.
The acquired data were imported into the software Spectronaut (Biognosys). As proteome background the human proteome data was selected from UniProt (www.uniprot.org) containing 20,374 entries. The processing settings were set as following: enzyme was trypsin, the minimum and maximum peptide length was set to 7 and 52 respectively, missed cleavages was set to 2. Carbamidomethyl for cystein was set as fixed modification and acetyl (Protein N-term) and oxidation of methionine was set as variable modifications. All settings regarding the library generation including tolerances, identification, filters, iRT calibration and workflow were set to factory defaults. For the relative quantification, the option Top N max 3 was taken meaning that for each protein the average of the 3 most intense identified peptides are taken to give the protein the quantitative value.
DIA-LC-MS/MS analysis
For the data independent acquisition (DIA) approach, the same nano LC-MS/MS setup as for the DDA acquisition was used. Each sample analyzed was mixed with an appropriate amount of iRT standard (Biognosys) peptides and 1 µg of each sample was subjected to the analysis. Full MS scans were acquired from 300-1100 m/z at a resolution of 60,000 (Orbitrap) using the polysiloxane ion at 445.12002 m/z as lock mass. The automatic gain control (AGC) was set to 5E5 and the maximum injection time to 20 milliseconds. Full MS scans were followed by 30 DIA windows acquired at a resolution 30,000 (Orbitrap) with an AGC set to 1E6, a maximum injection time of 60 milliseconds and nCE of 32 (HCD).
Analysis of DIA data
For the analysis of the samples acquired with nano-LC-MS/MS in DIA mode, the data was introduced to the Spectronaut software and analyzed with a library-based search. As library, the above created spectral library was used. Search and extraction settings were kept as standard (BGS Factory settings). As background proteome, same data were used as selected for the establishment of the spectral library.
Preparation of nuclear protein fraction from fibroblasts
Snap-frozen fibroblasts (3 controls and 3 patient samples) were processed utilizing the “Qproteome Cell Compartment Kit” (Qiagen; Cat No./ID: 37502): frozen cell pellets were dissolved with 1 ml of extraction buffer CE1 (+ protease inhibitor) and incubated for 10 min at 4 °C on a Thermomixer (Eppendorf). After centrifugation (1000 g for 10 min at 4 °C), supernatant was collected and pellets were dissolved in ice cold extraction buffer CE2 (+ protease inhibitor), followed by an incubation for 30 min at 4 °C (on a Thermomixer). After centrifugation (6000 g for 10 at 4 °C), the supernatant was collected in a new reaction tube and Benzonase was added to the pellets followed by an incubation for 15 min at RT. Afterwards 500 µl ice cold extraction buffer CE3 (+ protease inhibitor) was added and samples were incubated for 10 min at 4 °C. Insoluble material was pelleted by centrifugation with 6800 g for 10 min at 4 °C and the supernatant containing the nuclear proteins was collected and stored at -80 °C until further processing.
After thawing nuclear proteins fractions on ice, samples were mixed with ice cold acetone and incubated at -80 °C for 12 hours. After centrifugation (20.000 g for 20 min at 4 °C), supernatant was discarded, and the precipitated protein pellet was dried under a flow hood. Afterwards, 50 µl of 8 M Urea was added to dissolve the protein pellet at RT. After diluting the solution to 2 M urea with 10 mM ammonium bicarbonate buffer, pH 7.8 (ABC), a BCA-based determination of protein concentration was conducted according to the manufacturer’s instructions (Pierce BCA protein assay kit). Reduction and carbamidomethylation of the samples were carried out using 10 mM tris-(2-carboxyethyl)-phosphine (TCEP) for 30 min at 37 °C followed by application of 15 mM iodacetamide (IAA) for another 30 min at room temperature (RT). Samples were further diluted to 1 M urea using 10 mM ABC buffer adding trypsin (ratio 1:100) to warrant protein hydrolyzation over night at 37 °C. Next, the tryptic-digestion was terminated by adding 3 µl of 99% FA to decrease the pH to 2. Afterwards, samples were desalted using solid phase extraction with C18 filter cartridges, washed with 0.1% TFA and eluted with 80% ACN. Cleaned samples were dried by using a vacuum concentrator. Concentration was adjusted to 1 µg/µl with 0.1% TFA.
Microscopic studies
Fibroblasts
Fibroblasts derived from the Allgrove patient as well as from respective sex and age matched controls were grown on coverslips, washed three times with 1xPBS, fixed with 4% formaldehyde solution for 20 min at room temperature and afterwards washed thrice with PBS. After 200 µL BODIPY 493/503 (ThermoFisher Scientific, diluted 1:1000 in PBS) was added to each coverslip, samples were incubated in a climate chamber for 1 h at room temperature in the dark. The staining solution was removed, samples were washed thrice in 1xPBS and mounted onto a slide utilizing a drop of mounting medium (Prolong Gold Antifade reagent with DAPI, Invitrogen). After solidification of the mounting medium, samples were sealed with nail polish and stored at 6°C in the dark until further microscopic investigation.
Fluorescence measurements were performed on a modified Leica TCS SP8 CARS laser scanning microscope equipped with a 25x water immersion objective (Fluotar VISIR 25x/0.95 WATER). BODIPY fluorescence was excited at 488 nm and detected with a hybrid detector (Leica HyD, specifications can be found on the manufacturer’s website) in the range 495 - 600 nm. DAPI fluorescence was excited at 405 nm and detected with a photomultiplier tube ranging from 415 to 475 nm. For each image, both fluorescence measurements were performed sequentially. Fluorescence data were acquired as small 3D stacks with a resolution of 2048 x 2048 pixel (pixel size 227 x 227 nm) in X→Y direction and 9 - 12 layers in Z direction with a step size of 570 nm.
Data processing was performed using Matlab R2015a. For better comparability, measurements were preprocessed as following: for each image stack, the mean intensity was calculated in Z direction. For the resulting 2D image, background noise was reduced by setting all data points with intensities lower than 1% to this background threshold. To account for cosmic spikes, an upper intensity threshold was determined. Consequently, less than 0.1% of all data points above background threshold showed an intensity value higher than this threshold. Intensities of data points exceeding this upper intensity threshold were set to this value. Afterwards, the image was rescaled to full range (8 bit) between the two threshold values.
From the processed mean images, single cell images were selected using a manually defined irregular octagon. For each of these single cell images, an intensity histogram analysis was generated whereby histogram values were normalized to percentage values (to account for the different sizes of the single cell images). The total fraction of data points showing 50 % or more of the maximum intensity value was calculated for each single cell. At least 30 individual cells were evaluated per sample.
Muscle biopsy specimen
Cryosections for immunofluorescence studies were cut at 7 µm thickness, fixed in acetone for 5 min at –20 °C and air dried either to be stained directly or stored at –20 °C for subsequent staining, except of sections for anti-α-Dystroglycan staining, which were fixed for 1 minute at -20°C in an 1:1 mixture of ethanol/glacial acetic acid and then washed in PBS, not dried.
Following primary antibodies were used: anti-α-B-Cyrstallin (polyclonal rabbit, 1:200; Stressgen, Canada), anti-α-Dystroglycan (monoclonal mouse, 1:75; VIA4-1; Merck Millipore, Germany), anti-NCAM1 (monoclonal mouse, 1:100; Abcam), anti-HSP90 (polyclonal rabbit, 1:100; Genetex), anti-Ataxin 2 (polyclonal rabbit, 1:50; Abcam), anti-Periostin (polycloncal rabbit, 1:100; Abcam), anti-Synpatopodin-2 (M2 polyconal rabbit, 1:300 and HH9 monoclonal mouse, 1:2; both kindly provided by Peter F.M. van der Ven/Dieter O. Fürst). Primary antibodies were diluted in PBS and applied overnight at 4°C. Furthermore, fluorescence staining of Actin filaments was performed using the Phalloidin-iFluor 488 reagent (Abcam). Secondary antibodies used were Alexa 488 conjugated donkey anti-mouse IgG (1:400) and Alexa 555 conjugated donkey anti-rabbit IgG (1:800) (Molecular Probes, Invitrogen, Thermo Fisher Scientific, Germany). The secondary antibodies were diluted in 1x PBS and applied for one hour at room temperature. The sections were finally mounted in a Mowiol 4-88 (Calbiochem, Merck Chemicals, Germany) and glycerol mix in pH 8.5 Tris buffer with 0.1% DABCO (1,4-Diazabicyclo(2,2,2)octane; Sigma-Aldrich). Images were acquired with an AxioScope.A1 microscope using an Axiocam 503 color camera and ZEN 2.3 (blue edition) software (all Carl Zeiss Microscopy Ltd., Germany). Further image processing was performed with Adobe Photoshop CS6 Extended (Adobe Systems Inc., CA, USA).
Coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) measurements were performed on a modified Leica TCS SP8 CARS with an APE picoEmerald laser system. Ten micrometer thick sections were cut from cryo tissue blocks. The samples were stored at -80°C and thoroughly dried before measurements at room temperature. No further sample preparation was applied. The statistical evaluation is based on the 10 CARS images. A total of 103 muscle fibres were collected for the evaluation. For further details see supplemental document 1.