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Protein Preparation for MSMS protocol

Before you start (reagents, equipment, etc.)

  • 50 mM NH4HCO3 + 6M urea solution
  • Ethanol
  • BCA Assay (Pierce) - Kit that is purchased
  • 1.5 M Tris pH 8.8
  • 200 mM TCEP
  • 200 mM iodoacetamide
  • 200 mM diothiothreitol (DTT, freshly made or frozen)
  • pH test strips
  • Concentrated NaOH to adjust pH(5N)
  • 10% and 1% formic acid
  • Lys-C
  • Trypsin
  • Solvent A (60% acetonitrile + 0.1% trifluoroacetic acid)
  • Solvent B (5% acetronitrile + 0.1% trifluoroacetic acid)
  • ApoA1 (use for DIA analyses)
  • HPLC grade methanol
  • Lysis Buffer
  • Dry-ice (Health Sciences-J wing)
  • Wet ice (FTR Building)
  • Sonicater (Genome Sciences building for now)- broken one in Roberts Lab
  • Plate reader- Spectrophotometer (Genome Sciences building)
  • Disposable microplate
  • Pipettes and pipette tips
  • Snaptop centrifuge tubes
  • Centrifuge (FSH209 and FSH213)
  • Cold Speed Vacuum at 4C(Genome Sciences building)
  • Heating block at 37C (FSH209)
  • Macrospin columns (FSH 213)
  • pH meter for making 1.5M Tris pH 8.8 (FSH209)
  • Peptide Retention Time Calibration Mixture (DIA and SRM only, used for each sample on the mass spectrometer)

Ordering of Supplies

  • Trypsin: Life Technologies (20ug x 5 cat. 90057)

  • TCEP: Sigma (cat. C4706-2G)

  • Macrospin columns: The Nest Group (macrospin c18 columns, #SMM SS18V)

  • pH test strip: Cole Palmer (Hach pH Paper Test Strips, 0 to 14 pH Range; 100 Strips/Pk, cat: WU-05546-42)

  • Lys-C: Wako Chemicals (Lysyl Endopeptidase, cat: 125-02543)

  • Formic acid: Sigma (500mL HPLC formic acid, cat: 09676-500ML)

  • 2-Iodoacetamide: Sigma (IAA, cat: I1149-5G)

  • HPLC methanol: Fisher (1L HPLC methanol, cat:A4521)

  • BCA Protein Assay Kit: Fisher (BCA Protein kit, cat: PI23225)

  • Acetonitrile: Fisher Scientific (1L)

  • Urea: Fisher Scientific (2.5 kilos)

  • Ammonium Bicarbonate: Fisher Scientific (500g)

  • 100% Ethanol: Decon Laboratories Inc- Foxwest Sales (24 x 16oz bottles)

  • Dithiothreitol: Fisher Scientific (5 grams)

  • Tris(hydroxymethyl)aminomethane hydrochloride: Fisher Scientific (500g)

  • Triflouroacetic acid: Fisher Scientific (25 ml)

  • MS glass autosampler vials: ThermoFisher Scientific (Pack of 100, glass, clear, Convenience Kit, 9mm Wide Opening 1.5mLTotal Recovery Screw Vial with 10μL Reservoir, MSCERT5000-33TR)

  • Peptide Retention Time Calibration Mixture: ThermoFisher Scientific, 200 µL stock solution

To make reagents

50mM ammonium bicarbonate in 6M urea

Make only what you need for each sub-procedure as you must use within 24 hours of making it

To make a 20ml solution:

  • For ammonium bicarbonate (NH4HCO3)

    MW: 79.06g/mol

    (79.06g/mol)x(1mol/1,000mmol)x(50mM/1L)x(1L/1,000ml)x(1,000mg/1g)x 20ml = 79.06mg of NH4HCO3 for 20ml

    Dissolve 79.06mg NH4HCO3 into 10ml nanopure. Vortex. Add Urea (see below) before topping off to 20ml with nanopure.

  • For Urea add the following to the NH4HCO3 solution you just made:

    MW: 60.06g/ml

    (60.06g/mol)x(6mol/L)x(1L/1000ml)x 20ml= 7.21g Urea to add to the NH4HCO3

    Dissolve 7.21g Urea into the solution you just made. Vortex and add nanopure to reach 20ml.

200mM Iodoacetamide (IAA)

To make a 5ml solution (20ul required/sample). Make 200ul aliquots and store at -80C. Must keep in dark; wrap tubes in foil when ready to use.

MW: 184.96g/mol

(184.96g/mol)x(1mol/1,000mmol)x(200mM/1L)x(1L/1000ml)x(1000mg/1g)x 5ml= 184.96mg IAA for 5ml

Dissolve 184.96mg IAA in 4.00 ml nanopure. Vortex and add additional nanopure to reach 5.00 ml.

200mM Dithiothreitol (DTT)

To make a 5ml solution (20ul required/sample). Make 200ul aliquots and store at -80C.

MW: 154.25g/mol

(154.25g/mol)x(1mol/1,000mmol)x(200mM/1L)x(1L/1000ml)x(1000mg/1g)x 5ml= 154.25mg DTT for 5ml

Dissolve 154.25mg DTT in 4.00 ml nanopure. Vortex and add additional nanopure to reach 5.00 ml.

200mM tris(2-carboxyethyl)phosphine (TCEP)

To make a 5ml solution (2.5ul required/sample). Make 200ul aliquots and store at -80C.

MW: 286.65g/mol

(286.65g/mol)x(1mol/1,000mmol)x(200mM/1L)x(1L/1000ml)x(1000mg/1g)x 5ml= 286.65mg TCEP for 5ml

Dissolve 286.65mg TCEP in 4.00 ml nanopure. Vortex and add additional nanopure to reach 5.00 ml.

25mM NH4HCO3

To make a 25ml solution (800ul required/sample)-Must use within 24hrs of making it.

MW: 79.06g/mol

(79.06g/mol)x(1mol/1,000mmol)x(25mM/1L)x(1L/1,000ml)x(1000mg/1g)x 25ml = 49.4mg of NH4HCO3 for 25ml

Dissolve 49.4mg NH4HCO3 in 20ml nanopure. Vortex and add additional nanopure to reach 25ml.

1.5M Tris(tris(hydroxymethyl)aminomethane hydrochloride) pH8.8

To make 5ml of solution (6.6ul required/sample)

MW: 157.596g/mol

(157.596g/mol)x(1.5M/L)x(1L/1000ml)x 5ml= 1.182g Tris

Dissolve 1.182g of Tris in 3.00 ml nanopure. Vortex. Adjust pH with 5N NaOH to reach ph8.8 (vortex after every addition). Add more nanopure to reach 5.00ml.

Solvent A (60% acetonitrile + 0.1% trifluoroacetic acid)

To make 150ml (300ul required/sample)-can store on benchtop. (Minimize plastic use to make this solvent. Pour small amounts of reagents into beakers to pipette from and store working solution in glass bottle. You may pipette directly out of working bottle for procedure)

Add 90ml acetonitrile and 0.15ml trifluoroacetic acid to 59.85ml of nanopure

Solvent B (5% acetonitrile + 0.1% trifluoroacetic acid)

To make 150ml (500ul required/sample)-can store on benchtop. (Minimize plastic use to make this solvent. Pour small amounts of reagents into beakers to pipette from and store working solution in glass bottle. You may pipette directly out of working bottle for procedure)

Add 7.5ml acetonitrile and 0.15ml trifluoroacetic acid to 142.35ml of nanopure

3% acetonitrile + 0.1% formic acid for final step

To make 100ml (100ul required/sample). (Minimize plastic use to make this solvent. Pour small amounts of reagents into beakers to pipette from and store working solution in glass bottle. You may pipette directly out of working bottle for procedure)

Add 3ml acetonitrile and 0.1ml of formic acid to 96.9ml of nanopure


Sonication

Reagents required:

  • 50 mM NH4HCO3 + 6M urea solution (500ul/sample)
  • Ethanol (for dry-ice bath and sanitation)

Procedure:

For larvae:

  1. Add 500 µl 50 mM NH4HCO3 + 6M urea solution to each sample and homogenize larvae using pestle. After homogenization, spin samples down at 2000rpm for 5 minutes.
  2. Aliquot 150ul of supernatant to a new labeled tube. Save the remainder in the -80C.

For tissue:

  1. Split tissue sample in half using a clean razor blade. Place the half not being used for proteomics back in the -80ºC freezer.
  2. Add 5100 µl 50 mM NH4HCO3 + 6M urea solution to each sample and homogenize tissue using pestle. After homogenization, vortex tissue sample.

For both larvae and tissue:

  1. Make an ethanol dry-ice bath by getting a small plastic container and filling with ethanol. You will only need to dip one sample at a time. Slowly add small chunks of dry-ice until they are not dissolving very fast. This should be cold enough.

  2. Adjust sonicatior settings. Pipet an amount of water equal to that of the samples to be sonicated into a new centrifuge tube. Test sonication intensity by sonicating the water in that tube. Ideal intensity is the highest intensity at which no water sprays out of the centrifuge tube (ex. for Roberts Lab sonicator, use amplitude of 50% when sonicating samples that have a volume of 100 µL).

  3. Sonicate each sample for 10s and dip in ethanol + dry ice for 5s. Repeat twice for each sample (total of 3 times). When finished with a sample, put in dry ice bath, then wedge the tube into wet ice and move on to next sample.

  4. Between samples, rinse the sonicating probe with ethanol and nanopure. Dry with kim wipe.

  5. Aliquot 11ul of each sample to a new labeled tube for BCA assay. Save remainder for mini-trypsin digestion.

  6. Store all tubes at -80°C.


Protein quantification – BCA Assay (Pierce)

This needs to be done prior to Mini-Trypsin digestion.

Reagents required:

  • BCA working reagent is 50 parts BCA reagent A : 1 part BCA reagent B (600ul/sample and 600ul/standard)
  • BSA stock from kit (700ul for these 8 standards)
  • Lysis buffer is 50mM NH4HCO3 + 6M urea solution diluted 2:1 with nanopure (2.5ml total for these 8 standards)
  • 50mM NH4HCO3 (22ul/sample)

BCA standards (Genn's dilution curve):

Vial BSA conc. (µg/µl) Vol. lysis buffer (µl) Vol. BSA & dilution (ul)
B 1.5 125 375, stock
C 1.0 325 325, stock
D 0.75 175 175, B
E 0.5 325 325, C
F 0.25 325 325, E
G 0.125 325 325, F
H 0.025 400 100, G
I 0.000 500 N/A

Procedure:

  1. Make your 8 BCA standards in snaptop centrifuge tubes (see table). Make sure to vortex before using one standard to create your next standard.

  2. Create a table to describe the contents of the microplate wells.

  3. Make three replicates for each BCA standard. Pipette 10ul for each replicate into microplate well.

  4. Obtain your samples for the BCA assay (each tube has 11ul). Add 22ul of 50 mM NH4HCO3. Vortex and centrifuge down.

  5. Make three replicates for each unknown sample. Pipette 10 ul for each replicate into microplate well.

  6. Add 200ul of working reagent to every well- standards and unknown samples.

  7. Cover microplate and bring to Genome Sciences building. Log into computer attached to spectrophotometer and open SkanIt RE for Varioskan Flash 2.4.5 (there are two icons- pick bottom icon). Open new session and name it. Click on execution to connect. Use green and red arrows on bottom left hand side of screen to insert the microplate. The program will automatically incubate samples for 30 minutes at 37C, then shake to mix reagents, and then measure the absorbance at 562nm.

  8. When process is complete (takes about an hour), take a screenshot or photo of results and email it to yourself.

  9. Calculate the averages from the three replicates for both your standards and unknown samples.

  10. Subtract the average absorbance for the Blank Standard Replicates from the average absorbances of all the other standards and unknown samples. This is your blank-corrected absorbance.

  11. Create a scatter plot in excel for the standards by plotting the BSA concentration (ug/ul) on the Y-axis and the blank-corrected absorbances on the X-axis. Add a polynomial trendline and display the equation and R-squared value. This way you can easily plug in the X (absorbance) and calculate the Y (protein concentration).

  12. Because you diluted your sample 1:2 with 50 mM NH4HCO3 in 6M Urea, multiply the protein concentrations you calculated by 3.

  13. Then calculate how much volume is needed to have 100ug of protein in each sample. Because you will want 100ul to start the mini-trypsin digestion, calculate how much 50mM NH4HCO3 + 6M urea you will need to add to reach 100ul total volume.


Mini-Trypsin digestion

This method requires having 100ug of protein in each sample. You must also have 100ul volume to start. Therefore your concentration of protein is 100ug/ul.

Reagents required:

  • 50mM NH4HCO3 + 6M urea (determine amount needed from BCA assay)
  • 1.5M Tris pH 8.8 (6.6ul/sample)
  • 200mM TCEP (2.5ul/sample)
  • 200mM IAA (20ul/sample)- wrap tubes in foil when you get them out of -80C.
  • 200mM DTT (20ul/sample)
  • Lys-C (3.3ug/sample)
  • 25mM NH4HCO3 (800ul/sample)
  • HPLC methanol (200ul/sample)
  • Trypsin (3.3ug/sample)

Procedure:

  1. Turn on heating block in lab to 37C. Double check temperature as it heats up with another thermometer.

  2. Aliquot the equivalent of 100ug of protein from each of your samples into new labeled tubes.

  3. Add 50mM NH4HCO3 + 6M urea solution to reach final volume of 100ul.

  4. Add 6.6 µl of 1.5 M Tris pH 8.8

  5. Add 2.5 µl 200 mM TCEP and vortex.

  6. Test pH of samples to make sure they are still basic using pH test strips.

  7. Incubate samples 1 hour at 37°C.

  8. Add 20 µl of 200 mM iodoacetamide (IAA), vortex

  9. Incubate 1 hour, room temperature, in dark

  10. Add 20 µl 200 mM diothiothreitol (DTT), vortex

  11. Incubate 1 hour, room temperature

  12. For DIA analyses: Add ApoA1 to each sample (dilute 1.1 µl 2200 ng/µl stock in 48.9 µl of 50mM NH4HCO3 to make 50 ng/µl) to make 1:300 ratio of ApoA1:total protein. For 100 µg total protein in a sample, add 333 ng of ApoA1 by adding 6.7 µl of 60 ng/µl ApoA1 solution.

  13. Prepare LysC by adding 450ul nanopure to 2AU vial. This creates a 2ug/ul concentration of LysC. Make 20ul aliquots and store in -80C.

  14. Add 1.65ul LysC (1:30 enzyme:protein ratio) for 100ug of protein you have in each sample, vortex. (LysC cleaves at C-terminus of R and K, like trypsin, but works well in 6M urea.)

  15. Incubate 1 hour, room temperature

  16. Add 800 µl 25 mM NH4HCO3 and 200 µl HPLC grade methanol to each tube.

  17. Prepare the number of trypsin bottles needed (3.3ug/sample). Add 20 µl nanopure to each 20 ug bottle of trypsin and vortex lightly. This creates a 1ug/ul concentration of Trypsin.

  18. Add 3.3ul Trypsin (1:30 enzyme:protein ratio) for 100ug of protein you have in each sample, vortex.

  19. Incubate overnight at room temperature or for 4 hours at 37°C.

  20. Evaporate samples at 4°C to near dryness on speed vacuum. May take 10+ hours. Store at -80°C.

Desalting

Reagents required:

  • Solvent A = 60% acetonitrile + 0.1% trifluoroacetic acid (300ul/sample)

  • Solvent B = 5% acetonitrile + 0.1% trifluoroacetic acid (500ul/sample)

  • Final Solvent = 3% acetonitrile + 0.1% formic acid (100ul/sample)

  • Macrospin columns (Sample capacity: 0.03-300ug, Elution volume 50-150ul, Bed volume 300ul)

Other options for columns:

Column type Sample capacity(µg) Elution volume (µl) Bed volume (µl)
UltraMicro Spin 0.03-30 5-25 50
MicroSpin 0.05-60 10-50 100
MacroSpin 0.03-300 50-150 300
96-well MiniSpin 0.03-100 30-50 100
96-well MACROspin 0.03-300 60-150 300

Procedure:

  1. Reconstitute samples in 100 µl solvent B. Ensure pH2, if it isn’t, add 10 µl increments of 10% formic acid until pH is less than 2 (could take ~80ul of 10% formic acid). Test 1-2 samples and apply to the rest.

  2. Wash column: Add 200 µl solvent A to columns, spin for 2000 rpm 3 minutes (repeat 3 times for a total of 4 times). Discard remaining liquid everyother time to accomodate room for next round.

  3. Equilibrate column: Add 200 µl solvent B to columns, spin for 2000 rpm for 3 minutes (repeat 2 times for a total of 3 times). Discard remaining liquid everyother time to accomodate room for next round.

  4. Load protein on column: Add 100 µg of protein digest (1 sample per column). Spin at 3000 rpm for 3 minutes. Collect flow-through, put back on column and spin again. Your peptides are now in the columns. Transfer the remaining liquid to new tubes (just in case) and store at -80C.

  5. Wash salts through column: Wash columns with 200 µl solvent B, spinning at 3000 rpm for 3 minutes (repeat twice for a total of 3 times). Save remaining liquid at the bottom of each tube by transferring it to a new labeled tube (just in case) and store at -80C

  6. Elute peptides: Transfer columns to clean collection tubes. Add 100 µl solvent A, spin 3000 rpm for 3 minutes (repeat once for a total of 2 times). Your peptides are now in the liquid- do not discard!

  7. Evaporate samples to near dryness at 4C in speed vac (should take ~4hrs)

  8. Reconstitute peptides in 100 µl final solvent. Light vortex and centrifuge down. Store at -80°C.

PRTC Addition (DIA and SRM only?)

Reagents required:

  • Peptide Retention Time Calibration Mixture (0.5 pmol/µL stock solution from online order)

  • Final Solvent = 3% acetonitrile + 0.1% formic acid

  • Autosampler vials and clean centrifuge tubes for each sample

Procedure:

  1. Calculate amount of sample to be placed in autosampler vial: (µg to be injected/injection volume) x (final volume) / (sample concentration), i.e. ((1 µg/2 µl) x 15 µl) / (concentration)

  2. Calculate amount of PRTC to be added: (PRTC final concentration/injection volume) x (final volume) x (1/PRTC stock concentration), i.e. (50 fmol/2 µl) x (15 µl) x (1/200 fmol/µl)

  3. Dilute stock PRTC solution. Mix 20 µL 0.5 pmol/µL stock PRTC solution and 30 µL final solvent to get 50 µL 0.2 pmol/µL PRTC solution to add to samples. Scale up as needed for the number of samples to be used.

  4. In a clean centrifuge tube, pipet volume of sample calculated in Step 1 and 0.2 pmol/µL PRTC solution calculated in Step 2. Add as much final solvent as needed to get volume in the centrifuge tube up to 15 µL. Vortex gently.

  5. Pipet 15 µL sample + PRTC + final solvent solution into clean autosampler vial. Be sure to pipet slowly and without bubbles.

  6. Run samples on mass spectrometer immediately, or place in -80ºC freezer until ready to run.