Materials.
Urea and Thiourea, Bromophenol blue, Tris-base, Ammonium persulfate, Bradford protein assay reagent CHAPS, Iodoacetamide, DTT, E64, IPTG, Sodium carbonate, Sodium bicarbonate, TMB, DMSO, Aprotinin, Tryptone, SDS, Ni-agarose beads, Trypsin and Trypsin reaction buffer were from Sigma-Aldrich, USA. ASB-14-4 (tetradecanoylamidopropyl dimethyl ammonio-butane sulfonate, (Calbiochem, USA), Ampholyte (pH 3–10), IPG strips pH 3–10 and Mineral oil (biotechnology grade) (Bio-Rad), TEMED, Ethanol (Fisher Scientific, USA), Methanol (Merck, USA). Pencillin/streptomycin, Hybridoma serum free medium and Fetal bovine serum (FBS) and TOP10 from Invitrogen, USA. Protogel (National Diagnostics, USA), Amicon ultra-4 centrifugal filter unit, PVDF (Millipore, USA), Streptavidin-HRP conjugate Biotin (Thermo Scientific, USA), X-ray film and X-ray Casette (Kodak, India), Developer and Fixer (Premier, India), Acetonitrile and Ammonium bicarbonate (Fluka, USA), ESI-Q-TOF (Bruker, Germany), Ampicillin and Kanamycin (Hi-media, India), pCMV-XL5- gene construct of PFDN5α, CIP29, ECH1 and PRDX6 (Origene, USA). EcoRI, NotI, DNA ladder and T4 Ligase (TaKaRa, India), QIAquick kit (Qiagen, India), BL21 (Merk-Millipore, USA), pET28a vector (Merk-Millipore, USA), DH5α (ATCC, USA), Ammonium acetate, NaCl, Tween 20, Acetone, Yeast extract, Disodium hydrogen phosphate and HCl (Nice chemicals, India), Multiplate reader (BioTek, USA).
B-ALL Patient CSF Samples.
CSF samples from B-ALL patients were collected at CNS + ve (presentation/relapse positive for blast cells)/CNS–ve remission status with written informed consent according to institutional human ethics committee guidelines with approval (IEC-AIMS-2016 Dated 25-01-2016). CSF includes sequential samples from B-ALL patients of various age groups, both genders with different cytogenetics abnormalities and treatments (Table S1).
CSF Sample processing and biotinylation.
The CSF samples obtained from the same (sequential samples) and different patients were spun down (800g for 5 minutes at 4°C) to remove any cells and were kept as aliquots at -80°C until processed. CSF biotinylation were carried out with 20mM biotin (10% v/v) to CSF for 3.5 hours at room temperature and then stored at -20°C as per Menon et al 2011 (12).
Cell culture and cell lysate preparation.
The B-lymphoblastic JM-1 cell line was obtained from National Centre for Cell Sciences, Pune, India (originally from ATCC, USA). Cells were cultured in serum-free hybridoma medium containing 10% FBS and 1% penicillin/streptomycin at 37°C in 5% CO2 incubator as described in Xavier et al., 2010 (13). Cells were trypsin treated and spun down at 800g for 8 minutes. The pellet was PBS washed and lysed in 8M urea-2M thiourea solution with protease inhibitors E64 and Aprotinin. Further, presence of any particulate material in the lysate was dispersed by sonication and concentrated using Amicon ultra-4 centrifugal filter unit at 4500g for 45 minutes at 4°C and washed five times with 8M urea and then used for 2D PAGE profiling (13).
Protein estimation.
Protein estimation of concentrated cell lysate was assayed using Bradford reagent and absorbance was measured at 595nm using micro plate reader.
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).
2D-PAGE was performed essentially the same way as described in Xavier et al (13). Briefly, 200 µg total proteins from lysate sample was taken in 125 µL of rehydration buffer and rehydrated actively at 50V/20°C for 18 hours using IEF cell (Bio-Rad). After rehydration, the strip was subjected to IEF as described before (12, 13). This is followed by incubation in equilibration buffer (50mM Tris, pH 8.8, 6M urea, 30% glycerol, 2% SDS) with 0.3% DTT for 10 minutes. Subsequently, incubated with 4.5% iodoacetamide containing equilibration buffer for 10 minutes and subjected to SDS-PAGE. Proteins resolved in the gel were visualized by silver staining or used for western blotting (12).
2D-Far-western analysis using CSF.
2D-PAGE was performed using 200 µg total proteins as described above (13). For far western, biotinylated CSF sample (2.0µg concentration CSF proteins in 1ml of 5% gelatin at 4°C overnight) from patient was probed against lymphoblastic proteins on a 2D western blot. The blots were washed 3 times 10 minutes each in TBST (Tris Buffered saline + 0.2% Tween 20) and subjected to streptavidin-HRP staining (1 in 3000 dilutions with 5% milk in TBST). The chemiluminiscence signal following CSF reactivity to protein spots was captured on X-ray film. Subsequently, the CSF reactivity pattern obtained with CNS + ve at presentation was compared with sequential CSF samples reactivity pattern from the same patient collected at different periods of remission respectively. The procedure was repeated for other patient CSF samples. The sequential samples from patients varied in time frame from 3 weeks to 1.5 years. The intra and inter patient reactivity pattern analysis between three patients resulted in identification of 4 specific spots with consistent reactivity to CNS + ve/-ve CSF.
Quantification of CSF reactive spots on 2D blot by Image J software.
The CSF reactive spots corresponding to the spots marked in Fig. 1B was measured using the Image J software by making a selection on the spot. Then the value of the background of the same blot was subtracted from the spot value and the resulting quantity was considered as the value of the spot. The internal control spot corresponding to GAPDH which showed reactivity irrespective of CNS positivity and remission was also measured in the same way as mentioned above and the value corresponding to GAPDH was used for normalizing the value of these differentially reactive spots. The normalization was done by dividing value of the spot / Value of GAPDH multiplied by average value of GAPDH.
Mass spectrometric analysis of CSF reactive lymphoblastic proteins.
Based on the comparison of the CSF reactivity profile towards proteins of lymphoblastic origin on a 2D blot, the protein spots which showed differential reactivity to CSF (spot 1–4) were cut out from the Coomassie stained gel and de-stained with 100mM ammonium bicarbonate/Acetonitrile (1:1 vol/vol) solution and spun down (12). The gel piece was dehydrated by incubation for 30 minutes at room temperature in 100% acetonitrile with occasional vortexing. Then the gel piece was dried in vacuum and ice cold trypsin reaction buffer was added to cover the gel piece. The mixture was incubated at 370C for overnight till the gel piece becomes saturated with trypsin. The supernatant containing digested protein in the spot was collected. The proteins were identified by mass spectroscopy analysis using ESI Q-TOF mass spectrometry (12).
Cloning and expression of identified proteins using bacterial expression system.
The pCMV6-XL5-PFDN5α/CIP29/ECH1/PRDX6 was transformed to DH5α competent cells for plasmid amplification and subsequently purified by alkaline lysis method. The gene insert (PFDN5α/CIP29/ECH1/PRDX6) was excised from pCMV6-XL5 vector and ligated to pET28a bacterial expression vector. Subsequently transformed into BL21 strain and optimized the conditions for expression of protein/s.
Expression and purification of recombinant PFDN5α/CIP29/ECH1/PRDX6 from bacteria.
200ml culture of transformed BL21 (with pET28a-PFDN5α/CIP29/ECH1/PRDX6) was induced using 0.5mM IPTG at 37ºC for protein production. After 5 hours of induction, cells were pelleted down, lysed in lysis buffer of pH 8.0 (8M Urea, 50mM Tris-Base, 500mM disodium hydrogen phosphate) containing protease inhibitors, sonicated to remove the aggregates and centrifuged at 14000rpm for 5minutes. Supernatant containing the expressed protein was collected and subjected to Ni-NTA column purification as described by the manufacturer Sigma.
Validation and Translation of B-ALL CSF reactivity to PFDN5α for prognostication of CNS leukemia.
The purified recombinant PFDN5α/CIP29/ECH1/PRDX6 (2µg) was western blotted to PVDF membrane and probed with biotinylated CSF samples from B-ALL patients as before and the reactivity was quantified using Image J software. For ELISA, 300ng of purified recombinant PFDN5α protein coated on 96 well plates was incubated with 35ng of biotinylated CSF samples from the patients for 1hr at room temperature. Washed three times with TBST (TBS with 0.05% Tween 20) and incubated with Streptavidin HRP for 1hr at room temperature (1/7000 dilutions in 1% BSA). Washed in TBST for three times and incubated with HRP substrate TMB for 10 minutes. The reaction was stopped by adding 1N HCl and the color was measured at 450nm.
Statistical analysis
Unpaired two-tailed Students t-test assuming equal variances utilizing Graphpad Prism were carried out and statistical significance were set at p ≤ 0.05 and represented with star (*). Receiver operating characteristic curve (ROC) analysis was performed using SPSS software version 22.0 to identify a cut off value to predict CNS positivity in B-ALL patients.