Materials and Reagents

1.7 mL microtubes (Axygen, catalog number: GEN-MT-175-C)
15 mL centrifuge tubes (Axygen, catalog number: GEN-CT-15 mL)
Gloves (Tarsons, catalog number: 370110)
Pipette tips (Axygen, catalog numbers: T-300 [10 µL], T-200-Y [200 µL], T-1000-B [1,000 µL])
Lab Apron (Medifit)
Zeba spin desalting columns, 7K MWCO, 5 mL (Thermo Scientific, catalog number: 89892)
ActiveX Desthiobiotin-FP Serine Hydrolase (Thermo Scientific, catalog number: 88317)
High capacity streptavidin agarose (Thermo Scientific, catalog number: 20357)
Dimethyl Sulphoxide, DMSO (Sigma, catalog number: D2650)
Dithiothreitol, DTT (Sigma, catalog number: D9779)
Iodoacetamide (Sigma, catalog number: I6125)
MS grade trypsin from porcine pancreas (Sigma, catalog number: T6567)
Trifluoroacetic acid,TFA (Sigma, catalog number: T6508-10AMP-KC)
LC-MS grade water (J.T. Baker, catalog number: 39253)
LC-MS grade acetonitrile, ACN (J.T. Baker, catalog number: 9829-03)
Urea (MP Biomedicals, catalog number: 194857)
Phosphate-buffered saline (PBS, pH 7.4)
NP-40 (Sigma, catalog number: NP40S)
Ethylenediaminetetraacetic acid, EDTA (MP Biomedicals, catalog number: 194822)
Tris (Himedia, catalog number: MB029-500G)
Sodium chloride, NaCl (Sigma, catalog number: S3014-500G)
Protein extraction buffer (see Recipes)
Assay buffer (see Recipes)
Washing buffer (see Recipes)
Elution buffer (see Recipes)
Solvent A (see Recipes)
Solvent B (see Recipes)

Equipment

-80°C freezer (New Brunswick, Ultra-low temperature freezer, model-U410), for long term storage of the probe
-20°C freezer (ARCTIKO)
Single-channel pipette (Eppendorf, model: Research Plus, catalog numbers: K31824D [10 µL capacity], L24004D [100 µL capacity], L25129D [1,000 µL capacity])
Ice flaking machine (Model-CB 246 HC)
Ice bucket
UV-Spectrophotometer (Jasco, V-630Bio) for protein quantification
ROTOSPIN^TM – End to end Test Tube Rotator (Tarsons, model: 3071)
Water bath (Grant, SAP5, SL. no. UD1541001)
AccuBlock digital dry bath (Labnet, model: D1100)
Centrifuge (Eppendorf, model: 5418)
Centrifuge (Eppendorf, model: 5810R) with 15 mL adapter
Ultracentrifuge (Beckman Coulter, model: Optima XPN-100)
Shaker (New Brunswick Scientific, SL. No. SE24FG001727)
Concentrator plus (Eppendorf, Code: E5305000363)
Vortex mixer (Velp Scientifica, Code No. F202A0176)
Weighing balance (Sartorius, model: CP423S)
Thermo Scientific LTQ or LTQ Orbitrap XL Mass Spectrometer (for analysis)
Pre-column (75 µm × 2 cm; Nanoviper C18, 3 µm; 100Å)
Liquid chromatography analytical column (RSLC C18 3 µm; 50 cm × 75 µm; 100Å)

Procedure

Active site peptide enrichment
1. Aliquot 1 mg of protein in a 1.7 mL microtube, and make up the volume to 500 μL with assay buffer (described in Recipes section).
2. Add 10 μL (20 µM ) of desthiobiotin–FP serine hydrolase probe or DMSO (No probe control) as control and incubate at 37°C for 1 h.
3. Stop the reaction by adding 500 μL of 10 M urea. Next, add 10 μL of 500 mM DTT, and incubate the sample at 65°C for 30 min.
4. Allow the sample to cool down to room temperature (RT). Add 40 μL of 1 M iodoacetamide and incubate protected from light at RT for 30 min.
5. Desalt the protein using a Zeba spin desalting column, to reduce the urea to a suitable concentration for trypsin digestion (as per the manufacturer’s instruction).
6. Digest the desalted protein with 20 μg of MS grade trypsin at 150 rpm and 37°C for 7 h.
7. Affinity captur the labelled peptides on 75 μL of 50% high capacity streptavidin agarose, by incubating the matrix with the digested peptides at constant rotation at RT for 1 h.
8. Pellet the labelled peptides bound to streptavidin agarose beads by centrifugation at 1,000 × g at RT for 1 min .
9. Wash the beads three times with 1.5 mL of washing buffer (see Recipes). Mix the beads by briefly vortexing after the addition of the buffer. Centrifuge the sample at 1,000 × g at RT for 1 min to collect the beads, and carefully remove the supernatant without disturbing them. This precaution was followed for all the subsequent steps of washing.
10. Wash the beads three times with 1.5 mL of PBS buffer (pH 7.4, see Recipes) followed by three washes with 1.5 mL of LC-MS grade water.
11. Elute the captured peptides by adding 100 μL of elution buffer (see Recipes) and incubating the sample at RT for 3 min. This step was repeated two additional times and the eluted fractions were pooled together and lyophilized by vacuum concentration.
Mass spectrometry analysis of enriched probe targets
1. Suspend the lyophilized and labelled peptides in 0.1% TFA and inject them directly onto a Thermo LTQ Orbitrap coupled with an EASY nano LC1200 system (Agilent).
2. Set the column temperature to 35°C and limit the flow rate to 250 nL/min.
3. The LC solvent system consists of two mobile phases, such as Solvent A (see Recipes) and Solvent B (see Recipes).
4. Separate the peptides on the analytical column“[EASY SPRAY PREAMP (RSLC C18 3 µm; 50cm × 75µm; 100Å) silica column]”, by running a 180 min gradient of solvents A and B (Start with 10% B for 2 min, gradient 10–45% for 166 min, gradient 45–95% for up to 172 min).
5. Set the mass spectrometer in positive ion mode with the following parameters: precursor ion scanning in an orbitrap analyzer in the range of 375–1,700, at a resolution of 120,000, with a maximum ion injection time of 50 ms; high collision energy (CID) to 30% for the MS/MS spectra generation; MS²scan range (m/z) to 100–2,000, with a maximum ion injection time of 35 ms.

Data analysis

Peptide and protein identification
1. Convert raw data files to Mascot Generic File (MGF) files.
2. Search the MS/MS spectra data using a Mascot search engine (version 2.7.1.0).
3. The search parameters included: fixed modification of carbamidomethyl (57.02146 Da); variable modifications of acetyl (protein N-term) (42.01057 Da); oxidation of methionine (15.99492 Da); biotin: Thermo-88317 (S) (443.2913Da). Set enzyme specificity to Trypsin/P, and allow a maximum missed cleavage of 2.
4. Set the peptide mass tolerance to 10 ppm, fragment mass tolerance to 0.6 Da, ion score cut-off to 30, and the significance threshold to 0.05.
Statistical Analysis
Data are represented as mean ± standard deviation of three independent experiments. The significance of difference at p <0.01 was determined by one-way ANOVA with Tukey post hoc test using IBM SPSS Statistics software.

Recipes

Protein extraction buffer
50 mM Tris-Cl (pH 8.0)
150 mM NaCl
2.0 mM KCl
2.0 mM MgCl₂
10% glycerol
Autoclave at 121°C for 20 min and store at 4°C afterward.
Assay buffer
50 mM Tris-Cl (pH 8.0)
50 mM NaCl
10% glycerol
Autoclave at 121°C for 20 min.
Washing buffer
25 mM Tris-Cl (pH 7.4)
350 mM NaCl
1% NP-40
5% glycerol
Active site peptide elution buffer
50% ACN
0.2% of TFA
Dilute 20 μL of TFA with 5.0 mL of ACN and 5.0 mL of LC-MS grade water.
Solvent A
0.1% Formic acid in HPLC grade water
Solvent B
80% acetonitrile and 0.1% formic acid in HPLC grade water

Acknowledgments

The work was supported by the Department of Science and Technology, New Delhi, India, under the DST-INSPIRE Faculty Scheme (IFA14-LSPA28). AKD was supported by CSIR-UGC-JRF. M.L was sponsored by UGC-Rajiv Gandhi National Fellowship (RGNF) Award.

Competing interests

The authors declare no competing interests.

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