TITLE: Preparation of Proteins from Whole Body Larval Zebrafish Tissue for  Proteomics
AUTHORS: Abigail Henke

[DESCRIPTION]
This protocol describes the steps needed to prepare proteins from whole body larval zebrafish tissue for bottom-up shotgun proteomics via high resolution liquid chromatography mass spectrometry. Steps include solution preparation, sample collection, protein extraction, filter aided sample preparation, reduction, alkylation, enzymatic digestion via trypsin, and desalting steps using filter tips prepared in-house. This protocol results in a 1μg/μL solution of purified peptide samples.

[BEFORE_START]
Reserve any community equipment needed, perform equipment calibrations, and verify operational status of equipment prior to commencement of procedure. Pre-cool centrifuges and pre-heat heat blocks. 

Read the full procedure and determine pause points before starting. 

[GUIDELINES]
This protocol is for the extraction, filter-aided sample preparation (FASP), reduction, alkylation, digestion, acidification, and desalting of proteins from whole body larval zebrafish (Danio rerio) tissue for bottom-up shotgun data-independent acquisition (DIA) proteomics.

[STEPS]
SECTION: Solution Preparation For Protein Extraction Steps
1. Phosphate Buffered Saline  (PBS, 1X, 1L)
Obtain a clean 1L glass media bottle, 1000mL graduated cylinder, sodium chloride (NaCl), potassium chloride (KCl), disodium phosphate (Na2HPO4), potassium phosphate (KH2PO4), a mass balance capable handling milligram scale measurements, a pH probe and pH balancing solutions (standard 1M HCl and NaOH), and an ultrapure water source. 

Record the manufacturer, lot number, date opened, and date of expiration for all reagents.
Measure out the following and add to the 1L glass media bottle:
8.0 g of NaCl. Record actual mass. 
0.200 g KCl. Record actual mass. 
1.44 g  Na2HPO4. Record actual mass. 
0.245 g KH2PO4. Record actual mass. 
Record the model and identification of the balance used.   

Record source and purity of ultrapure water. Measure 900 mL of ultrapure water using the graduated cylinder and add to the 1L bottle. Mix to dissolve. 

Measure the pH of the solution with the pH probe and adjust to pH 7.4 ± 0.1 with 1M HCl or 1M NaOH. Record pH. Record model and identification of the pH probe used.

Bring solution up to 1000 mL using a graduated cylinder. Return solution to 1L media bottle. 

Label: Phosphate buffered saline, [Initials], date of preparation, date of expiration (2 years from preparation date at ambient room temperature), storage location, and storage temperature. 
SECTION: Solution Preparation For Protein Extraction Steps
2. Tris-HCl Stock, (0.1M, pH 7.8, 50mL)
Obtain a clean 100mL glass media bottle, 100mL graduated cylinder, tris-hydrochloride (Tris-HCl) a mass balance capable handling milligram scale measurements, a pH probe and pH balancing solutions (standard 1M HCl and NaOH), and an ultrapure water source.  

Record the manufacturer, lot number, date opened, and date of expiration for all reagents.
Measure out the following and add to the 1L glass media bottle: 
0.788 gof Tris-HCl. Record actual mass. 
Record the model and identification of the balance used.   

Record source and purity of ultrapure water. Measure 40 mL of ultrapure water using the graduated cylinder and add to the 100mL bottle. Mix to dissolve. 

Measure the pH of the solution with the pH probe and adjust to pH 7.8 ± 0.1 with 1M HCl or 1M NaOH. Record pH. Record model and identification of the pH probe used.

Bring solution up to 50 mL using a graduated cylinder. Return solution to 100mL media bottle. 

Label: Tris-HCl 0.1M Stock, pH:[actual], [Initials], date of preparation, date of expiration (6mo from preparation date at ambient room temperature), storage location, and storage temperature.
SECTION: Solution Preparation For Protein Extraction Steps
3. Dithiothreitol (DTT) Stock, (1M, 10 mL) 
Obtain a clean 15mL polypropylene falcon tube, 10mL graduated cylinder, dithiothreitol (DTT, C4H10O2S2), a mass balance capable of handling milligram scale measurements, an ultrapure water source, 16 1.5mL microcentrifuge tubes, 1000μL pipette and appropriate tips. 

Record the manufacturer, lot number, date opened, and date of expiration for all reagents.
Measure out the following and add to the 15mL falcon tube: 
1.5425 g of DTT. Record actual mass.  
Record the model and identification of the balance used.   

Record source and purity of ultrapure water. Measure 10 mL of ultrapure water using the graduated cylinder and add to the tube. Cap and mix to dissolve.  

Open the 1.5mL microcentrifuge tubes and aliquot 600 µL of DTT into each. Cap. Label and store in the dark at -80C.  
 

Label: Dithiothreitol (DTT) Stock, 1M,  [Initials], date of preparation, date of expiration (3mo from preparation date at -80C), storage location, and storage temperature. 
SECTION: Solution Preparation For Protein Extraction Steps
4. Lysis Buffer (0.05M Tris-HCl, 0.05M DTT, Protease Inhibitors, 10mL, enough to prepare 24 samples) 
Obtain a clean 15mL polypropylene falcon tube, 10mL graduated cylinder, Tris-HCl Stock, Dithiothreitol (DTT) Stock aliquot, Roche cOmplete Protease Inhibitor Tablet (04693116001), an ultrapure water source, and 1000μL pipette and appropriate tips. 

Record the manufacturer, lot number, date opened, and date of expiration for all reagents.
Measure out the following and add to the 15mL falcon tube:  
5 mL of 0.1M Tris-HCl stock reagent 
500 µL of DTT Stock, thawed in the dark 
1 Tablet of Roche cOmplete Protease Inhibitor. 

Record source and purity of ultrapure water. Measure 10 mL of ultrapure water using the graduated cylinder and add to the tube. Cap and mix vigorously with a vortex to dissolve. Place solution on ice, do not store past 24hrs.  

Label: Lysis Buffer Working Solution, [Initials], date of preparation, date of expiration (24hr from preparation date). 
SECTION: Solution Preparation For Protein Extraction Steps
5. Sodium Dodecyl Sulfate (SDS) Stock, (10%, 10mL) 
Obtain a clean 15mL polypropylene falcon tube, sodium dodecyl sulfate (SDS, CH3(CH3)11OSO3Na), a mass balance capable handling milligram scale measurements 10mL graduated cylinder, and an ultrapure water source. 

Record the manufacturer, lot number, date opened, and date of expiration for all reagents.
Measure out the following and add to the 15mL falcon tube:  
1.0 g of SDS reagent. Record actual mass.
Record the model and identification of the balance used.

Record the manufacturer, lot number, date opened, and date of expiration for all reagents.
Measure out the following and add to the 15mL falcon tube:  
1.0 g of SDS reagent. Record actual mass.
Record the model and identification of the balance used.

Record source and purity of ultrapure water. Bring SDS up to 10 mL with ultrapure water using the graduated cylinder and add to the tube. Cap and mix gently with a vortex to dissolve, but avoid frothing. Store solution at Room temperature.
 

Label: SDS Stock, [Initials], date of preparation, date of expiration (6mo from preparation date at ambient room temperature), storage location, and storage temperature. 
SECTION: Tissue Collection
6. Obtain 2mL low protein binding (lo bind) microcentrifuge tubes, an ice bucket with crushed ice, 200μL pipette and appropriate tips, plastic dropper pipettes, a dewar of liquid nitrogen, and liquid nitrogen personal protective equipment (protective cold gloves, eye protection, lab coat, forceps). 

 

 


Place the container of larval fish into the ice bucket for 5 min minutes to slow fish swimming speeds.

 



SECTION: Tissue Collection
7. Count and individually collect larval fish with a disposable dropper pipette, placing fish in the 1.5mL lo-bind tube. Cap tube and place on ice until all fish gravitationally pellet at the bottom. Remove as much water as possible with the dropper pipette. 
SECTION: Tissue Collection
8. Depress 200μL pipette to the first stop. Place the tip at the bottom of the fish pellet and slowly release plunger to remove all of the water without disturbing the fish. Cap tube and label. 
SECTION: Tissue Collection
9. Gently lower the tube into the liquid nitrogen with forceps to snap freeze the sample. Repeat steps 7 and 8 until all samples are collected. 
SECTION: Tissue Collection
10. Remove sample collection tubes from the liquid nitrogen using forceps. Place in a sample box and store samples between -20 °C  and -80 °C until extraction. 

SECTION: Protein Extraction
11. Obtain wet ice,  1000-, 200-, 20-, and 10μL pipettes with appropriate tips, a mass balance capable of making milligram scale measurements, a heat block, ultrapure water source, benchtop centrifuge capable of accommodating at least 24 samples, prepared Lysis Buffer,  prepared SDS Stock, 1 spare 2mL lo bind microcentrifuge tube, 1.5mL lo bind microcentrifuge tubes, a Scilogex (110-120V-60Hz, Speed = 8000-3000r/min, watts= 160W) homogenizer, a probe sonicator, ring stand with crimp attachment, Kimwipes, a waste beaker, and and spray bottle of 70% ethanol. 

Record equipment identification data, reagent preparation dates, and reagent expiration dates.

Cool benchtop centrifuge down to 4 °C prior to use.

Turn on heat block to 90 °C. Record actual temperature. 

SECTION: Protein Extraction
12. Remove collected tissue samples from storage and thaw on wet ice. This procedure can accommodate 24 samples / day. 

Place lysis buffer on ice. 
SECTION: Protein Extraction
13. Wash thawed tissue samples with 500 µL 1X PBS. Centrifuge samples for 10 sec at 4C using the “quick spin” function.
Gently pipette off supernatant without disturbing the tissue pellet or losing tissue. It is not critical to remove all buffer at this step. 
SECTION: Protein Extraction
14. Repeat wash step. Pipette off as much supernatant as possible without disturbing the pellet. Return samples to ice. 
SECTION: Protein Extraction
15. Tare balance with an empty 2mL tube from the same lot used to collect samples. Measure and record the approximate mass of protein samples in each tube. 
SECTION: Protein Extraction
16. Perform a 1:20 dilution of tissue with Lysis Buffer. 

Volume of Lysis Buffer to add (uL) = (wet weight of tissue (mg) × 20)

If samples + Lysis Buffer are below the 200uL marking on the 2mL tube, add lysis buffer to 200uL final volume.

Record volume added to each sample tube. 
SECTION: Protein Extraction
17. Clean Silogex homogenizer with 70% ethanol and assemble. 

Place homogenizer probe 1-2mm from the bottom of the sample tube.

Homogenize samples with a homogenizer on level 4 speed for 30 seconds moving the probe up and down slowly to thoroughly homogenize sample. Promptly return each sample to ice. 

Rinse probe in ultrapure water, spray with 70% ethanol, and wipe with a Kim wipe. 

Repeat for all samples. 

Clean homogenizer by disassembling and cleaning parts in 70% ethanol. Allow to dry and reassemble. 
SECTION: Protein Extraction
18. Add 10% SDS stock to each tube until it comprises 2%. 

Volume of SDS Stock to add (uL) = (Volume of lysis buffer added (uL) × 2) ÷ 10 

Do not place samples on ice as SDS will precipitate out of solution. 
SECTION: Protein Extraction
19. Place samples in a 90 °C heat block for5 minwith weighted top to prevent samples from popping their lids.
SECTION: Protein Extraction
20. Clean sonicator instrument and secure microtip probe. Program sonicator to operate in 15 second bursts with 15 seconds of delay for a total of 1 minute sonication at 60hz. 

Set up ring stand and crimp holder capable of securely holding sample tubes inside the sonication cabinet. 

 


Secure a sample tube in the crimp holder. Lower the sonicator probe 1-2mm below the surface of the sample without touching the tube itself. Close door and run sonication program. 

Wipe probe with 70% ethanol and a Kim wipe between samples. 

Repeat for all samples.
SECTION: Protein Extraction
21. Return samples to a 90 °C heat block for 5 min, then cool for 15 min at ambient room temperature.

SECTION: Protein Extraction
22. Centrifuge samples at 14000 rpm, 45 min, 4 °C .

SECTION: Protein Extraction
23. Remove samples from centrifuge and return to ice without disturbing the dark pellet (unlysed cells, insoluble protein, pigments, cell membranes, waste). 

Pipette off supernatant (no portion of dark pellet) and place in a labeled 1.5mL lo-bind tube. This is the extracted protein and should be kept on ice while in use and stored at -80 °C following BCA protein quantitation.  

SECTION: Protein Extraction
24. (Optional steps 24-27 to increase total protein yield) 
Resuspend the un-lysed pellet in each tube with 96 µL Lysis Buffer and 24 µL of 10% SDS stock. Vortex well to resuspend.

SECTION: Protein Extraction
26. Remove samples from heat block (turn off heat block) and place in centrifuge at 14000 rpm, 15 min, 4 °C .
SECTION: Protein Extraction
27. Pipette off supernatant and add to the labeled protein tube without disturbing or sucking up remaining dark pellet. 
Discard pellet and raw tissue storage tube.

Immediately store extracted protein samples at -80 °C or proceed with BCA quantitation. 
SECTION: Solution Preparation for Filter-Aided Sample Preparation, Reduction, Alkylation, Digestion, and Acidification
28. ABC Buffer (50mM Ammonium Bicarbonate, 100mL)
Obtain a clean 100mL glass media bottle, 500mL graduated cylinder, ammonium bicarbonate (ABC, HCO3), a mass balance capable handling milligram scale measurements, a pH probe and pH balancing solutions (standard 1M HCl and NaOH), and an ultrapure water source. 

Record the manufacturer, lot number, date opened, and date of expiration for all reagents.
Measure out the following and add to the 100mL media bottle
1.9764 g of ABC. Record actual mass.  
Record the model and identification of the balance used.   

Record source and purity of ultrapure water. Measure 400 mL of ultrapure water using the graduated cylinder and add to the tube. Cap and mix to dissolve.  

Measure the pH of the solution with the pH probe and adjust to pH 8.5 ± 0.1 with 1M HCl or 1M NaOH. Record actual pH. Record model and identification of the pH probe used. 

Bring solution up to 500 mL with ultrapure water using the graduated cylinder. Return to media bottle. 

Label:  ABC Buffer 50mM, [Initials], date of preparation, date of expiration (2yr from preparation date at ambient room temperature), storage location, and storage temperature. 
SECTION: Solution Preparation for Filter-Aided Sample Preparation, Reduction, Alkylation, Digestion, and Acidification
29. Urea (UA) Buffer, (0.01M Tris-HCl, 2M Urea, pH 8.5, 25mL) 
Obtain a clean 50mL polypropylene falcon tube, 50mL graduated cylinder, Urea (NH2CONH2), prepared Tris-HCl Stock, a mass balance capable of handling milligram scale measurements, an ultrapure water source, a pH probe and pH balancing solutions (standard 1M HCl and NaOH), 1.5mL microcentrifuge tubes, 1000μL pipette and appropriate tips.

Record the manufacturer, lot number, date opened, and date of expiration for all reagents.
Measure out the following and add to the 15mL falcon tube: 
3.003 g of Urea. Record actual mass.  
Record the model and identification of the balance used.   

Add 2.5 mL of 0.1M Tris-HCl stock reagent. Cap and Vortex well to mix. 

Record source and purity of ultrapure water. Measure 15 mL of ultrapure water using the graduated cylinder and add to the tube. Cap and vortex vigorously to dissolve.  

Measure the pH of the solution with the pH probe and adjust to pH 8.5 ± 0.1 with 1M HCl or 1M NaOH. Record pH. Record model and identification of the pH probe used.

Bring solution up to 25mL with Ultra-Pure water using the graduated cylinder. Return to falcon tube and mix well. 

Label:  Urea (UA) Buffer, [Initials], date of preparation, date of expiration (24hr from preparation date at room temperature), storage location, and storage temperature. 
 

SECTION: Solution Preparation for Filter-Aided Sample Preparation, Reduction, Alkylation, Digestion, and Acidification
30. Iodoacetamide (IAA) Stock (1M, 0.01M Tris-HCl, 2M Urea, 1mL) 
Obtain iodoacetamide (IAA, C2H4INO), prepared Urea (UA) Buffer, a mass balance capable of handling milligram scale measurements, an ultrapure water source, 1.5mL microcentrifuge tubes, 0.6mL microtubes, 1000- and 100μL pipettes with appropriate tips. 

Record the manufacturer, lot number, date opened, and date of expiration for all reagents.
Measure out the following and add to a 1.5mL microcentrifuge tube: 
0.18496 g of IAA. Record actual mass.  
Record the model and identification of the balance used.    

Add 1 mL of UA buffer to the tube. Vortex until mixed.  

Aliquot solution into 0.6mL tubes, 60 µL  in each tube.

Label: IAA Stock, [Initials], date of preparation, date of expiration (1yr from preparation date at -80 °C), storage location, and storage temperature. 

 

SECTION: Solution Preparation for Filter-Aided Sample Preparation, Reduction, Alkylation, Digestion, and Acidification
31. Iodoacetamide (IAA) Working Buffer, (0.05M IAA, 0.01M Tris-HCl, 8M Urea, 1mL)
Obtain a 1.5mL microcentrifuge tube, prepared UA buffer, prepared IAA Stock, 1000- and 100μL pipettes with appropriate tips. 

Record the date opened and date of expiration for all reagents.

Thaw aliquot of IAA by wrapping in tinfoil and allowing to come to ambient room temperature prior to use. 

Add 50 µL of IAA Stock to a 1.5mL microcentrifuge tube.  Discard remaining stock. 

Add 950 µL of UA buffer to the tube, vortex to mix. Wrap in foil until use.


 


Label: IAA Working Buffer, [Initials], date of preparation, date of expiration (24hr from preparation date at ambient room temperature).  

SECTION: Solution Preparation for Filter-Aided Sample Preparation, Reduction, Alkylation, Digestion, and Acidification
32. Priming Solution (50:50 Methanol: Water, 50mL)
Obtain a 50mL falcon tube, 100mL graduated cylinder, Methanol (CH3OH), and an ultrapure water source. 

Record the manufacturer, lot number, date opened, and date of expiration for all reagents.
Measure 25 mL of Methanol. 

Record source and purity of ultrapure water. Measure 25 mL of ultrapure water using the graduated cylinder and add to the tube. Cap and mix.  

Label: Priming Solution, [Initials], date of preparation, date of expiration (1yr from preparation date at ambient room temperature), storage location, and storage temperature. 
SECTION: Solution Preparation for Filter-Aided Sample Preparation, Reduction, Alkylation, Digestion, and Acidification
33. Sodium Deoxycholate Solution (1% DOC, 0.01M Tris-HCl, 10mL)
Obtain a 15mL falcon tube, sodium deoxycholate (DOC, C24H39NaO4), prepared Tris-HCl Stock, a mass balance capable of handling milligram scale measurements, 10mL graduated cylinder, 1.5mL microcentrifuge tubes, and an ultrapure water source. 

Record the manufacturer, lot number, date opened, and date of expiration for all reagents.
Measure out the following and add to a 1.5mL microcentrifuge tube: 
0.1 g of DOC. Record actual mass.  
Record the model and identification of the balance used. 

Record source and purity of ultrapure water. Measure 9 mL of ultrapure water using the graduated cylinder and add to the tube. Cap and mix to dissolve.  

Add 1 mL of Tris-HCl Stock to the tube. Vortex until mixed.

Aliquot solution into 1.5mL tubes, 1 mL  in each tube.

Label: 1% DOC Solution, [Initials], date of preparation, date of expiration (6mo. from preparation date at -80 °C), storage location, and storage temperature. 

 


SECTION: Solution Preparation for Filter-Aided Sample Preparation, Reduction, Alkylation, Digestion, and Acidification
34. Trifluoroacetic acid (TFA) Buffer (5% TFA, 20mL)
Obtain a 100mL glass media bottle, 50mL graduated cylinder, 1000μL pipette with appropriate tips, 1mL glass Trifluoroacetic acid (TFA, CF3CO2H) ampule, and an ultrapure water source. 

 

Record the manufacturer, lot number, date opened, and date of expiration for all reagents. 
Prepare this solution inside of a ventilation hood.
Gently break TFA ampule and pipette 1 mL  contents into the 100mL glass media bottle. 

Record source and purity of ultrapure water. Measure 19 mL of ultrapure water using the graduated cylinder and add to the bottle. Cap and mix.  

Label: TFA 5% Solution, [Initials], date of preparation, date of expiration (1yr from preparation date at ambient room temperature), storage location [inside of an acid storage cabinet], and storage temperature.
SECTION: Filter-Aided Sample Preparation
35. Obtain 30kD Amicon Centrifugal filter units, microcentrifuge, thermomixer fitting 24 samples, ultrapure water source, ice bucket with ice, 1000-, 200-, 100-, 20-, and 10μL pipettes and appropriate tips, 15mL falcon tube, 1.5mL lo bind microcentrifuge tube, sequencing grade modified porcine trypsin (Promega, frozen, 5-20ug, cat#V5113), prepared DOC Solution, prepared DTT stock, prepared IAA working solution, prepared priming solution, prepared UA buffer, prepared NaCl solution, prepared TFA Buffer, and prepared ABC buffer. 

Preheat thermomixer to 50 °C.
SECTION: Filter-Aided Sample Preparation
36. Thaw samples on wet ice. Thaw frozen Trypsin on wet ice.

Record source and purity of ultrapure water. Collect 10 mL into a 15mL falcon tube.

Label 30kD Amicon Centrifugal filter units and accompanying 2mL tubes with sample names.  Pipette 200 µL of ultrapure water into each filter unit. Visually inspect each filter unit for leaks or damage. 

Spin filter units at 14000 x g, 15 min, 25 °C . 
Visually inspect filter units to ensure liquid is passivating through. If solution does not passivate through the filter, discard and prime another filter unit before proceeding.
SECTION: Filter-Aided Sample Preparation
37. Pipette  200 µL of Priming Solution into each filter unit. 

Spin filter units at 14000 x g, 15 min, 25 °C . 

Decant remaining solution from filter, but do not allow filters to dry out.
SECTION: Filter-Aided Sample Preparation
38. Using calculations from previously performed BCA assay, add  50 µg of protein sample to each respectively labeled filter unit.

Add  200 µL of UA buffer to each unit. 
Pipette to mix 15-20 times.

Spin samples at 14000 x g, 30 min, 25 °C .

 

SECTION: Filter-Aided Sample Preparation
39. Add 100 µL of ABC Buffer to each filter unit.

Spin samples at 14000 rpm, 20 min, 25 °C . 


 

SECTION: Filter-Aided Sample Preparation
40. Repeat Step 38. Discard eluate. 
SECTION: Protein Reduction
41. Transfer samples to the pre-warmed thermomixer. 

Add20 µL of DTT stock to each unit and shake for 1 minon thermomixer at  . 

Incubate in the dark for 30 min at50 °C. 
SECTION: Protein Alkylation
42. Add 11 µL of IAA Working Buffer to each filter unit. Shake at for 1 min and incubate samples in the dark for 30 minat 50 °C. 

Pre-heat thermomixer to 37 °C .

SECTION: Protein Digestion
44. Wash filter units with 100mL ABC buffer. 

Spin samples at 14.000 x g, 30 min, 25 °C . 

Discard eluate.
SECTION: Protein Alkylation
43. Spin samples at14.000 x g, 20 min, 25 °C . 
SECTION: Protein Digestion
45. Transfer samples back to the thermomixer. Using the volume markings on the side of the filter unit, add ~ 30 µL of prepared DOC Solution to each filter unit until you reach a total volume of 60μL.

Add3 µL of Trypsin to each tube (a total of 1ug Trypsin, Ratio of 1:50). Mark trypsin usage on the lid, thawing no more than 3 times total before disposal. 
SECTION: Protein Digestion
46. Shake samples at   for1 minand incubate samples at 37 °C for 900 min in the thermomixer shaded with a heated top. 

SECTION: Protein Digestion
47. Add 1.5 µL Trypsin to each tube and mix at   for1 min . 
Incubate for 180 min at 37 °C in the thermomixer shaded with a heated top. 
.

SECTION: Protein Digestion
48. Spin samples 14000 rpm, 7 min, 25 °C .
Add 100 µL of NaCl solution, and mix in thermomixer at   for 15 min. 

SECTION: Protein Digestion
49. Transfer filter units to a clean collection tube. 

Invert filters and collect supernatant by spinning at 4.000 x g, 5 min, 25 °C .
SECTION: Protein Acidification
50. Acidify samples by adding 80 µL of 5% TFA Solution (to a final concentration of 2% TFA) to each sample. Vortex to mix.
SECTION: Protein Acidification
51. Spin samples at 4000 rpm, 5 min, 25 °C and transfer the supernatant to labeled 1.5mL lo bind microcentrifuge tubes. 

Store samples at -80 °C or immediately proceed with sample desalting. 

SECTION: Solution Preparation for Peptide Sample Desalting
52. Solution A (100% Methanol, 5mL) 
Obtain a 15mL falcon tube, 5mL serological pipette, pipette aid, and HPLC-grade Methanol (CH3OH). 

Record the manufacturer, lot number, date opened, and date of expiration for all reagents.
Measure out the following and add to the 15mL falcon tube: 
5 mL HPLC grade methanol. Cap tube. 

Label: Solution A, [Initials], date of preparation, date of expiration (1 week at ambient room temperature), storage location [flammables cabinet], and storage temperature. 
SECTION: Solution Preparation for Peptide Sample Desalting
53. Solution B (60% Acetonitrile, 39.9% Water, .1% Formic Acid, 5mL) 
Obtain a 15mL falcon tube, 5mL serological pipettes, pipette aid, 10μL pipette and appropriate tips, HPLC-grade Acetonitrile (ACN, CH3CN), HPLC-grade water, and HPLC-grade Formic Acid (or >95% purity, HCOOH).  

Record the manufacturer, lot number, date opened, and date of expiration for all reagents.
Measure out the following and add to the 15mL falcon tube:  
3 mL HPLC-grade acetonitrile.
2 mL HPLC-grade water. Use a 10μL pipette to remove 5 µL and add
5 µL Formic acid. 

Cap and vortex to mix. 

Label: Solution B, [Initials], date of preparation, date of expiration (1 week at ambient room temperature), storage location (flammables cabinet), and storage temperature. 
SECTION: Solution Preparation for Peptide Sample Desalting
54. Solution C (.1% Formic Acid in Water, 8mL) 
Obtain a 15mL falcon tube, 10mL serological pipettes, pipette aid, 10μL pipette and appropriate tips, HPLC-grade water, and HPLC-grade Formic Acid (or >95% purity, HCOOH).
  
Record the manufacturer, lot number, date opened, and date of expiration for all reagents.
Measure out the following and add to the 15mL falcon tube:  
8 mL HPLC-grade water. Use a 10μL pipette to remove 5 µL and add
5 µL Formic acid. 
Cap and vortex to mix. 

Label: Solution C, [Initials], date of preparation, date of expiration (1 week at ambient room temperature), storage location, and storage temperature. 
SECTION: Solution Preparation for Peptide Sample Desalting
55. Solution D (80% Acetonitrile, 19.9% Water, .1% Formic Acid, 8mL)
Obtain a 15mL falcon tube, 10mL serological pipettes, pipette aid, 10μL pipette and appropriate tips, HPLC-grade Acetonitrile (ACN, CH3CN), HPLC-grade water, and HPLC-grade Formic Acid (or >95% purity, HCOOH).   

Record the manufacturer, lot number, date opened, and date of expiration for all reagents.
Measure out the following and add to the 15mL falcon tube:  
6.4 mL HPLC-grade acetonitrile.
1.5 mL HPLC-grade water. Use a 10μL pipette to remove 5 µL and add
5 µL Formic acid. 

Cap and vortex to mix.   

Label: Solution D, [Initials], date of preparation, date of expiration (1 week at ambient room temperature), storage location (flammables cabinet), and storage temperature.
SECTION: Solution Preparation for Peptide Sample Desalting
56. Formic Acid Solution (0.1% Formic Acid in Methanol) 
Obtain a clean 100mL glass media bottle, 100mL graduated cylinder, 200μL pipette and appropriate tips, HPLC-grade Methanol (CH3OH), and HPLC-grade Formic Acid (or >95% purity, HCOOH). 

Record the manufacturer, lot number, date opened, and date of expiration for all reagents.
Measure out the following and add to the 100mL glass media bottle:  
99.9 mL HPLC-grade Methanol. 
100 µL Formic acid. 
Cap and mix. 

Label: Formic Acid Solution, [Initials], date of preparation, date of expiration (1 week at ambient room temperature), storage location (flammables cabinet), and storage temperature.
SECTION: Filter Tip Preparation
57. Obtain 3M Empore 2240 High performance Extraction Disks, 200μL unfiltered pipette tips, 10μL unfiltered pipette tips, 2.0mL lo-bind tubes, 1.5mL lo-bind tubes, an 18 gauge flat tip syringe needle, compressed air duster with fine tip, and a 3mm Philips head screwdriver. 
SECTION: Filter Tip Preparation
58. Label and prepare a 2.0mL and 1.5mL tube for each sample by puncturing a hole in the center of each tube cap with the 3mm Philips head screwdriver. 

 

Widen hole as necessary until a 200μL unfiltered pipette tip can be fitted snugly into each tube with the tip end inserted to approximately the 1.0mL volume line marked on side of each tube.   


 

The 2.0mL tube will serve as the wash collection tube. 
The 1.5mL tube will serve as the sample collection tube. 
The inserted 200μL tip in each will serve as the filter tip protector sheath. 
SECTION: Peptide Sample Desalting
60. Obtain ice bucket with ice, prepared filter tips, prepared Solution A,  prepared Solution B, prepared Solution C, prepared Solution D, prepared Formic Acid Solution, Waters MassPREP™ Enolase digestion standard (SKU: 186002325), a microcentrifuge (capable of accommodating 24 samples simultaneously), a Speed-Vap solvent evaporation apparatus (capable of accommodating 24 samples simultaneously) or equivalent instrument, Nanodrop or peptide quantification kit, glass autosampler tubes with re-sealing caps (compatible with liquid chromatography autosampler to be used), 200-, 100-, 20-, and 10μL pipettes with appropriate tips. 

Record date of preparation, date of expiration, and lot number of all reagents where possible. 

 


Pre-heat Speed-Vap to 65 °C and turn on vacuum pumps.  

SECTION: Peptide Sample Desalting
61. Thaw samples on ice.
Determine peptide concentration via NanoDrop. Record peptide concentrations and determine the volume of each sample needed for 30 µg of peptide.
SECTION: Peptide Sample Desalting
62. Activate column by adding 75 µL of Buffer A to the 10μL filter tip seated in the filter protection sheath of the wash tube. Spin at 4000 x g, 30 s, 25 °C. Liquid should be passivated through the filter, but the filter should not dry between steps.

SECTION: Peptide Sample Desalting
63. Activate column by adding 75 µL of Buffer D. Spin at 4000 x g, 30 s, 25 °C . Liquid should be passivated through the filter, but the filter should not dry between steps.
SECTION: Peptide Sample Desalting
64. Activate column by adding 75 µL of Buffer C. Spin at 4000 x g, 30 s, 25 °C . Liquid should be passivated through the filter, but the filter should not dry between steps.
SECTION: Peptide Sample Desalting
65. Repeat column activation steps. 

Visually examine each filter tip and collection tube to ensure that filter layers have not been lost and that solvent is passivating through. If a tip is defective, discard it, prepare a new one, and activate as described above.

Decant solution from the wash tube and discard. Place column back in the wash tube sheath protector.

 Label each filter tip and tube for its respective peptide sample.
SECTION: Peptide Sample Desalting
66. Based on calculations from the BCA protein quantification in step 27, add 30 µg of peptide to each respectively labeled filter tip.

Spin at 4000 x g, 2 min, 25 °C  to bind protein to the filter. 
SECTION: Peptide Sample Desalting
67. Draw up eluate from the collection tube and reapply to the filter tip. 
Spin at 4000 x g, 2 min, 25 °C to bind more protein to the filter. 
SECTION: Peptide Sample Desalting
68. Repeat step.
SECTION: Peptide Sample Desalting
69. Wash tip with 75 µL of Buffer C to remove salts from filter-bound proteins.  

Spin at 4000 x g, 1 min, 25 °C . 
SECTION: Peptide Sample Desalting
70. Transfer filter tip to the protector sheath of the collection tube. Discard wash tubes.
SECTION: Peptide Sample Desalting
71. Add 50 µL of Buffer B to the column and spin at 4000 x g, 1 min, 25 °C . Keep eluate. 
SECTION: Peptide Sample Desalting
72. Add 50 µL of Buffer D to the column and spin at 4000 x g, 1 min, 25 °C . Keep eluate. 
SECTION: Peptide Sample Desalting
73. Repeat step 70. Discard filter tips and keep eluate in labeled collection tubes.
SECTION: Peptide Sample Desalting
74. Cap collection tubes and place into the pre-warmed Speed-Vap (the hole in the lid where the filter tip was previously placed will allow solvent evaporation). 

Run the Speed-Vap in 5-10 minute intervals closely monitoring the volume of each sample until each is reduced from 150μL to a final volume of 5-10μL.  

Remove samples from speed-vap and turn off apparatus.   


 

SECTION: Peptide Sample Desalting
75. Individually, use a 10μL pipette with appropriate tips to draw up sample from the lo bind collection tube, estimate the volume of peptide, and transfer to a labeled glass autosampler vial. 

Bring total volume in the autosampler vial up to 29μL with Formic Acid Solution. 
SECTION: Peptide Sample Desalting
76. Add 1uL of Waters MassPREP™ Enolase digestion standard to each sample. 
Cap and vortex well to mix.

 
 
SECTION: Peptide Sample Desalting
79. Immediately proceed with liquid chromatography tandem mass spectrometry data acquisition or store samples at -80 °C until analysis. 

SECTION: Peptide Sample Desalting
77. Prepare an intrastudy quality control (QC) sample by transferring 1 µL from each sample into a single fresh autosampler vial. Label as  instrastudy QC with date of preparation.

Cap and mix.
SECTION: Peptide Sample Desalting
78. Prepare an enolase intra/inter laboratory standard at 25 fmol/μL. 
SECTION: Protein Extraction
25.     Return samples to a 90 °C heat block for 5 min, then cool for 15 min at ambient room temperature.

SECTION: Filter Tip Preparation
59. Prepare the filter tip by placing a 10μL unfiltered pipette tip in the protector sheath of the wash collection tube. 

Using the 18 gauge flat tipped syringe needle, punch 3 holes through the Empore filter disk, collecting the punched circles inside the hollow syringe needle. Place the end of the needle in the 10uL tip and with the fine applicator of compressed canned air, release a puff down the needle to dislodge the filter paper punches and deposit them snugly in the 10uL tip to create the filter column.