Thiol-free reducing agents in electrophoretic separations and FASP proteolytic digestions for the analysis of metal-binding proteins

Graphical abstract


Method details
Two protocols are detailed for the analysis of metal-protein complexes in biological samples, based on methods recently demonstrated for kidney cytosolic proteins from rats treated with platinumbased chemotherapeutic drugs. The described procedures include either TCEP-based rSDS-PAGE [1], or TBP-based rOFFGEL-IEF in combination with a FASP tryptic digestion [2], followed by identification by nLC-ESI-LTQ-MS/MS.
Prior to both protocols, a sample preparation step should be performed in order to obtain cytosolic protein fractions with the highest Pt to protein ratio from the metallodrug-exposed kidney by size exclusion chromatography (SEC)-ICP-MS.
Quick Start Bradford protein assay (Bio-Rad) Quadrupole ICP-MS equipped with a Meinhard nebulizer, a fassel torch, and an impact bead quartz spray chamber cooler by a Peltier system. Amicon Ultra-4 mL centrifugal filter (Millipore) with a nominal cut-off of 3 kDa. Procedure 1. Place the kidney tissue (about 0.250 g) from a rat treated with a pharmacological dose of a metalbased chemotherapeutic drug, such as oxaliplatin, in a Petri dish on ice and mince the tissue with a scalpel.! Caution: Always wear laboratory gloves when handling and preparing solutions, samples, apparatus, etc. to prevent contamination of samples and solutions (i.e. skin keratins). This caution is to be extended to all the protocols described later. 2. Place the tissue in a tube and add 3 mL of pre-chilled buffer solution. To minimize proteolysis, add 12.5 mL of the protease inhibitor cocktail and always keep the tube on ice during steps 2-4.
The FASP procedure allows including previous reduction and alkylation steps in addition to proteolysis, ensuring the preservation of the metal-protein complexes. The limited time that proteins remain in contact with the reducing agent, either TBP or even DTT, during FASP could be a key factor for its extraordinary performance on the digestion of metal-protein complexes. 3. Homogenize for a minimum of 15 min using a tight-fitting Teflon pestle attached to a power drill (set to >1000 r.p.m.) by slowly stroking the pestle up and down, taking 15 s per stroke and 30 s to complete a down and up cycle. 4. Inspect the homogenate; if intact tissue is still apparent, re-homogenize for an additional 1 min.! Caution: Wear safety glasses and a face shield, as the glass tube may shatter if too much pressure is applied or if the drill is moved at an angle other than vertical. 5. Decant the homogenate into an appropriate-size centrifuge tube and place into a pre-cooled rotor and centrifuge at 15,000 Â g for 40 min. 6. Decant and save the cytosolic supernatant on an ice-bath.~Critical step: all the previous preparative steps should be performed at 4 8C to minimize the risk of species degradation or transformation. Inert atmosphere is not needed. Possible species oxidation could be considered by setting oxidized methionines as variable modifications in Sequest searches.&Pause point: The supernatant can be kept frozen at À80 8C until required.
7. Inject the kidney cytosolic fraction through a 0.22 mm PVDF filter to the SEC column.
8. Collect fractions every minute (0.8 mL each). 9. Determine the Pt content in each fraction by ICP-MS (monitoring 195 Pt isotope, channels per AMU: 10 and integration time: 0.6 ms), and the protein content by Bradford assay. 10. Identify the fractions with the highest Pt to protein ratio, which are selected for further analysis. 11. For their preconcentration and clean-up, the pool of ten identical selected SEC fractions was ultrafiltered through an Amicon Ultra-4 mL, 3 kDa cut-off filter. Collect the retentate. Critical step: avoid boiling samples with the reductant in order to preserve the integrity of the metal-protein complexes.
4. Load the sample (a total protein content of 50 mg) into the gel wells. Start electrophoresis at constant current (12 mA per gel until samples were stacked an then the current was increased to 20 mA per gel until the end of the separation). 5. Wash the gels with Milli-Q grade water for 20 min and incubate the gel in the fixing solution for 1 h in an oscillating shaker. 6. Stain the gel with colloidal Coomassie Blue for 1 h. 7. Wash the gel twice with 20 mL of Milli-Q grade water for 1 h per wash. 8. Excise protein bands from the gels with a scalpel. 9. Wash the gel slices for at least 1 h in 500 mL of 50mM NH 4 HCO 3 . Discard the wash. 10. Wash the gel slices in 500 mL of 50% acetonitrile/50 mM NH 4 HCO 3 with shaking for 1 h. Discard the wash. Cut the gel band into 1 mm 2 pieces and transfer to a 500 mL Protein Lo-Bind tube (Eppendorf). Critical step: Make sure that the gel slices stay wet with the wash solution to facilitate cutting and transfer.
11. Add 50 mL of acetonitrile to shrink the gel pieces. After 10-15 min, remove the solvent and dry the gel slices in a centrifugal evaporator. 12. Re-swell the gel pieces with 50 mM NH 4 HCO 3 containing 12.5 ng/mL of trypsin and keep them in ice during reswelling (as required, typically add 10-20 mL). Once the gels have completely reswollen, add 50 mM NH 4 HCO 3 to cover the gel pieces (around 10-20 mL). Cap the tubes tightly and cover with parafilm to avoid evaporation. Incubate at 37 8C overnight ($16 h) with gentle agitation using a thermomixer at 300 r.p.m.
Critical step: gel pieces need to stay wet during the digestion. 13. After completing the digestion, collect supernatants and transfer them to a Lo-Bind Eppendorf, keeping them at 4 8C. Next, extract the peptides remaining in the gel with 30 mL of 2% formic acid by vortexing, and incubate for 30 min at RT. Pool the extracted peptides with the original supernatant.
14. Finally, add 30 mL of a solution containing 50% acetonitrile and 0.1% formic acid, and incubate for another 30 min at RT. Pool the extraction solution with the previous ones, and evaporate samples in a vacuum centrifuge to dryness. Store the dry peptides at À80 8C until required, within a few months.

For protein identification, analysis by nLC-ESI-LTQ-MS/MS (or a similar LC-MS/MS platform) is
proposed. Resuspend the dried peptides in 10 mL of buffer A and sonicate for 10 min in an ultrasonic bath. Then start the analysis as follows: Inject aliquots of 5 mL, using a 20 mL loop and a pick-up method, and load on a trap-column at a 3 mL min À1 flow rate using buffer A as mobile phase. Reverse the flow at 200 nL min À1 to elute and transfer the preconcentrated peptides to a reverse phase microcapillary analytical column. Elute peptides applying a three-step gradient: 5-15% B linear for 5 min, 15-40% B linear for 40 min and 40-80% B linear for another 15 min, holding the system at 80% B for 10 min. The end of the column was connected to a stainless steel nano-bore emitter for spraying and coupling with the LTQ. The spray voltage was set at 1.70 kV. Peptides were scanned and fragmented using a triple play scan method, consisting on acquisition of full enhanced MS scans in the positive ion mode, over the m/z range 400-1600, followed by zoom scans and further full enhanced MS/MS, acquired in profile mode, of the three most intense peaks in the full MS scan. CID activation of ions was applied in MS/MS experiments, with 35% relative collision energy and 30 ms activation time, being isolation width of the precursor ions set to 4. Dynamic exclusion was enabled with a repeat count of 1, using a 180 s exclusion duration window. For data analysis, spectra were assessed with the Xcalibur Qual Browser software (Thermo Scientific). MS/MS spectra search on NCBI protein databases using SEQUEST and MASCOT allowed the identification of proteins. The search was performed against a rat (Rattus norvegicus) NCBI database.

B1. TBP-based rOFFGEL-IEF
The following instructions assume the use of an Agilent 3100 OFFGEL Fractionator. Similar devices can also be used.

Procedure
1. For subsequent OFFGEL-IEF fractionation, add 2.88 mL of focusing buffer and 0.72 mL of Milli-Q grade water (3.6 mL final volume) to the preconcentrated cytosolic fraction (the retentate fraction from step 11 (sample preparation procedure)) and vortex. With respect to the original OFFGEL protocol for protein separation [4], DTT was replaced by TBP.
Critical step: Do not add thiourea (used in the original OFFGEL protocol) to the focusing buffer, in order to preserve the integrity of the metal-protein complexes, such as cisplatin, as was earlier reported [5]. OMIX Tips C 18 , 100 mL (Agilent Technologies).
NanoLC system coupled to an ESI-LTQ-MS/MS instrument. Buffer A: 2% Mass Spec-grade acetonitrile, 0.1% formic acid in mass spec-grade water. Buffer B: 0.1% formic acid in mass spec-grade acetonitrile. Database search engine such as Sequest or Mascot (Matrix Science) for protein identification.