Proteomics Protocols

• Trypsin digestion - long method (higher coverage, includes reduction and alkylation)
• Isolation of Tissue Dendritic Cells by Enzymatic Dissociation
• Optimization of Live Salmonella Infection in BMM
• In Vivo Expansion of Dendritic Cells using Flt3L-Secreting Melanoma
• Isolation of BMDM from mouse femurs
• Concentration of Collected Supernatant
• Mass Spec Analysis of Serum Isolated from Mice Infected with Salmonella

Trypsin digestion - long method (higher coverage, includes reduction and alkylation)

1. Excise protein spot/band, chop into ~1mm cubes, and wash 2X in 1 ml H20.

If coomassie stain:

a) Add 100ul H20, 15 min, pull off H20, add 40 ul 50/50 ACN/H20, 15 min

b) Pull off soln, add 40 ul ACN, incubate until white and sticky

c) pull off soln. Add 40 ul 100 mM NH4HCO3, 5 min.

d) Add 40ul ACN to make 1:1 solution, 15 min-can sonicate.

e) pull off soln, (if dye still present, repeat from b until dye is gone, or ~3 times total).

f) speed vac till dry (when you tap the tube see chunks fly)

If silver stain start here after step 1:

Dehydrate in ~100ul CH₃CN for approx. 15 min. Remove CH₃CN and SpeedVac until dry (15-30 min.)

2. Cover gel pieces with 10 mM DTT in 100 mM NH₄-HCO₃. Reduce proteins for 1 h at 56 C.

3. Cool to room temp, remove DTT solution and add equal volume of 55 mM iodoacetamide in 100 mM NH₄-HCO₃. Incubate for 45 min in dark place at room temp.

4. Wash gel pieces with 50-100 ul aliquots of 100 mM NH₄-HCO₃ for 5 min. Add equal volume CH₃CN for 15 min. Remove solution and repeat.

5. Remove liquid phase and speedvac (15-30 min.)

6. Reswell gel pieces at 4 ^oC for 45 min in buffer containing trypsin and 50 mM NH₄-HCO₃. (Approx. 5 uL/mm² gel). The gel pieces should just be covered (~40 ul).

Suggested amount of trypsin is 12.5 ng/uL (500ng/40ul) of buffer for proteins that have been silver stained. Digest overnight at 37°C (or at least for 3 h).

7. Centrifuge gel pieces and collect supernatant.

8. Further extract peptides by one change of 20mM NH₄-HCO₃ for10 min. followed by 2X volume CH₃CN for 15min. (centrifuge then collect).

9. Extract by addition of 5% acetic acid for 15 min. followed by addition of 2X volume CH₃CN for 15 min. (centrifuge then collect). Repeat 2 more times. (Can use sonicator water bath during CH₃CN extraction)

10. Dry sample down in speedvac until desired volume has been reached.

Note NEVER use more than 1 ug trypsin per sample for MS analysis.

Isolation of Tissue Dendritic Cells by Enzymatic Dissociation

Materials:

DC Media, 500 ml of RPMI + 10% heat-inactivated FBS + 5 ml 100x L-Glutamine + 5 ml 100x pen-strep + 3.6 ul ?-mercaptoethanol.

Hanks Buffered salt solution

Liberase Blendzyme 3 (Roche 11814176001 7 mg, resuspend 3.5 mg/ml PBS, 100 ul aliquots)

EDTA solution 0.5 M (Gibco 15575-038)

FACS Buffer, 2% FBS in 500 ml of PBS

MACS Running Buffer, 500 ml PBS + 8.3 ml of 35% BSA (Sigma A7979-50ml) + 2 ml 0.5 M EDTA

MACS Rinsing Buffer, 500 ml PBS + 2 ml 0.5 M EDTA (above autoMACS)

Anti-mouse CD11c Microbeads (Miltenyi Biotech 130-042-201) or pan-DC microbeads.

Procedure:

1. Preparation: spray TC hood with 70% EtOH, sterilize one pair of forceps, warm media and Hanks, thaw 100 ul Liberase per spleen, prepare two sets of 6 well (non-TC treated) or Petri dishes: one with cold media and store on ice, the other with 3.5 ml of warm Hanks/spleen and store in TC incubator.
2. Sacrifice mice by approved euthanasia techniques.
3. Remove spleen (or other organ, nodes, etc), place in media and cover to keep sterile. Perform rest of procedure in TC hood.
4. Add 100 ul of Liberase per 3.5 ml Hanks or 500 ul Collagenase D + 4.5 ml Hanks per well of a 6 well non-TC treated dish. This can be scaled down to 1 ml per well of a 24-well dish for lymph nodes (Note: use Liberase when LPS contained in the Collagenase may be a problem, i.e. for arrays or in vitro DC activation.)
5. Take up 3 ml of the Hanks/Liberase mixture into a 3 ml syringe, attach a 25 gauge 5/8" needle, and inject Hanks into the spleen to balloon the organ. This is not necessary for lymph nodes.
6. Tease the spleen into very small pieces using needle and forceps or mince using small dissection scissors (1 mm² pieces).
7. Incubate at 37°C 5% CO₂ for 25 minutes.
8. Add 70 ul of 0.5 M EDTA per 3.5 ml and mix (20 ul per ml) to dissociate DCs from T cells.
9. Incubate at 37°C for 5 minutes (spleen should be spindly in texture)
10. Place a 70 mm cell strainer on a 50 ml Falcon tube and using a 10 ml pipette, pipette spleen up and down until dissolved.
11. Pass through strainer and wash dish and strainer with 40 ml of Hanks. Remove 10 ul and count, mix with 10 ul of Turks stain (not Trypan), and count while cells are centrifuging. Expect 100-200 million splenocytes from normal mice and more this after injection with B16-Flt3L melanoma (therefore may need to dilute before counting if thre is Flt3L-induced splenomegaly).
12. Centrifuge at 350 xg for 10 minutes.
13. (Optional RBC lysis: resuspend pellet in 1 ml room-temperature ACK lysis buffer, incubate 1 minute, then add at least 9 ml of complete media or FACS buffer or Hanks. Pellet cells at 350 xg for 10 minutes. RBC lysis is not necessary prior to autoMACS purification).
14. Remove 1x10e6 cells for FACS staining to know extent of Flt3L expansion. Important: keep cells on ice.
15. Resuspend the cells in 460 ul of MACS buffer and add 40 ul of ?-mouse CD11c microbeads/spleen (in Carrie RÕs brown fridge in MACS box; double volumes for expanded spleen).
16. Incubate at 4^oC for 20 minutes, protected from light.
17. After incubation, fill tube with FACS buffer (50 ml) and spin down for 10 minutes at 1200 rpm.
18. Resuspend in 500 ul MACS running buffer (not FACS buffer), pipette up and down 10 times.
19. Filter the sample using 40 um cell strainers into a 50 mL conical and rinse 3x with 0.5 ml running buffer.
20. Can load 2x10e8 labeled cells and 4x10e8 cells per autoMACS column (max). If necessary, divide filtered cells between tubes for multiple autoMACS runs.
21. AutoMACS: use posseld for CD11c beads. Run QRinse between samples (of the same type) if multiple runs are required to avoid overloading columns.
22. Bring positive fraction to 10 ml with FACS buffer, pooling cells from multiple runs. Remove 10 ul to count after mixing with 10 ul Trypan.
23. Centrifuge cells at 350 xg for 10 min.
24. Resuspend cells at 1 million per ml in warm DC media and stimulate.
25. Save 2x10e5 cells prior to stimulation to measure purity by FACS.
26. Collect 5x10e5 cells from unstimulated and stimulated cells at final time point to confirm activation by FACS, saving supernatants from each time point as well for ELISA (want to confirm that unstimulated cells are not activated by the isolation procedure).

AutoMACS Magnetic Cell Sorter

1. Turn cell sorter on.
2. Exchange empty bottle on right side of machine with AutoMACS running buffer and cap empty bottle to keep sterile.
3. Perform column exchange function, if necessary (columns must be exchanged every 2 weeks). Refer to cheat sheet located on top of instrument for instructions.

If column exchange is not necessary, run "Clean" program. Make sure empty 50 ml conical is on intake port and empty waste conicals are placed under elution ports.

4. Immerse intake port in 70% EtOH with dry with Kimwipe. Spray elution ports with 70% EtOH with dry with Kimwipe.
5. Prepare AutoMACS for separation
1. Place 15 ml conicals under the appropriate elution ports ("pos 2" and "neg 1" for posseld positive selection)
2. Load sample onto intake port
3. Prepare pipette with 500 µl running buffer
4. Choose "Separation" function on screen
1. Highlight "Posseld" for positive double sort and click OK
6. Add running buffer when sample uptake nears end.
7. Perform quick rinse ("Qrinse") between samples of the same type.
8. Choose "Sleep" function after completing final separation
9. Immerse intake port in EtOH and replace empty bottle on right side
10. Turn off power and return Running buffer to TC fridge.
11. Refer to FACS cell sorting protocol to check purity (FACS should be run w/in 3 days of sort)

Solutions for AutoMACS:

All solution prep done in hood. Solutions need to be filtered if solution components are unfiltered.

1. Running Buffer- 2mM EDTA, 0.5% BSA in PBS. For 500 ml total:

500mL PBS

2 mL 0.5 M EDTA

7.1 mL 30% BSA

2. Rinsing Buffer- 2mM EDTA in PBS. For 500 mL total:

500mL PBS

2 mL 0.5 M EDTA

(often bottles above autoMACS)

FACS to Assess DC Purity

1. For FACS staining, stain cells live and always keep on ice and spin in refrigerated centrifuge (<2 000 rpm).
2. Resuspend cells (pre- and post-autoMACS) to stain a minimum of 1x10e5 per 100 ul per condition. Resuspend 1x10⁶ cells in 500 ul FACS buffer + 1 ul 2.4G2 purified Ab.
3. Mix and incubate on ice for 15 min.
4. Prepare 2x Ab dilutions while cells are blocking (DOUBLE the concentrations listed below).
5. Aliquot 50 ul of each cell type/condition per well/tube
6. After blocking, add 50 ul of 2x antibody mixes per condition.
7. Incubate primary Ab on ice for 20-30 min (protect from light).
8. Wash twice with FACS buffer (200 ul washes, 1500 rpm 5 min 4 C pellet).
9. Make up 1:1000 dilution of propidium iodide (PI). 5 ul per 5 mls.
10. Resuspend samples with 200 ul PI-containing FACS buffer and resuspend control cells with 200 ul of regular FACS buffer.
11. After staining, store on ice, protected from light.
12. Look at cells by flow cytometry and collect 20 000 Ð 50 000 events.
13. Conditions (stain splenocytes with a-f and AutoMACS-enriched cells with e-f).
1. Unstained (no PI)
2. Unstained
3. 1:200 CD11c-PE
4. 1:200 CD8?-FITC
5. 1:200 CD11c-PE + 1:200 CD8a-FITC
6. 1:200 rat IgG2a-FITC + 1:200 hamster IgG-PE

Unexpanded DCs are 1-3% of total splenocytes) and can reach up to 30% following in vivo expansion.

Optimization of Live Salmonella Infection in BMM

052808 Katie S.

Day 1

Replate cells at specific density in media with NO ANTIBIOTICS

1. CFU- seed 2 columns (16wells) of a 96 well plate w/ 4.15 x 10⁴ cells per well.
2. Immunofluorescence- seed 5 wells of a 24 well plate 2.31 x 10⁵ cells per well.

In a snap cap tube grow up a 1ml culture of ATCC 14028s WT and ATCC 14028s GFP (Kanamycin) in 37°C Shaker

Day 2

Spin down 1ml of bacteria

Resuspend in 1ml of media

Take OD of a 1:10 dilution (should be OD 4-5). OD 4= 4.0 x 10⁹ cfu/ml. Use this # to calculate the MOI

Infect Macs with bacteria at specific MOI

1. CFU- MOI of 200, 133, 89, 59, 40, 26, 17, 12. 1^st tube has 1.5mls at MOI 200. Take 500ul from this tube and add to 1ml of media (2^nd dilution = MOI 133) Continue the dilution curve until MOI 12

Immunofluorescence- MOI 200, 89, 40, 17 with Salmonella GFP and spin down for 5min

Incubate for 30 min at 37°C

Aspirate, wash 3X PBS

Add media with 15ug/ml Gentamicin (MCSF)

Incubate to given time point. (3.5 hrs for a total of 4hr stimulation)

Process Cells

1. CFU
1. Wash 3X to remove Gentamicin
2. Lyse macs in 200ul PBS 1% Triton, put on ice
3. Plate 10ul spot dilutions, 8 dilutions total (2 plates)
4. Incubate ON 30 C (downstairs)
2. Immunoflurescence-
1. Wash 1X

Day 3

Determine optimal MOI that results in 1-2 bugs/mac

1. CFU Ð count colonies on bacterial plates.
2. Immuno- view CS under confocal microscope.

In Vivo Expansion of Dendritic Cells using Flt3L-Secreting Melanoma

Culture of B16-Flt3L cells and transplantation

1. Grow cells in 10%FCS/DMEM. Cells grow adherent. Cells should be passaged every 2-3 days. No selection is needed. Do not keep cells in culture for more than needed. Once you have enough cells to injection, freeze the ones left in the cryotube.
2. To harvest cells, use 0.25% trypsin. Wash cells twice in complete DMEM to remove any trypsin.
3. Resuspend them in 0.1 to 0.2 ml (for one mouse) of PBS or HBSS no sera for injection.
4. Inject 4-6 x 10⁶ cells in C57BL/6 mice or any I-A^b haplotype mice. All mice should develop a tumor in 12-14 days. In any other haplotype inject 30-60 x 10⁶ cells. Not all mice will develop a tumor depending on the strain. Inject using an insulin syringe and a 25G needle s.c. in the back.
5. You can start checking for tumor development after one week. Euthanize the mice when the tumor is 1-2 cm, depending how many DCs you need.
6. Proceed with Isolation of Tissue DCs by Enzymatic Dissociation protocol, AutoMACS Purification prototol, and assess DC purity by flow cytometry.

Protocol provided by R. Steinman lab.

Reference: Dranoff G, Jaffee E, Lazenby A, Golumbek P, Levitsky H, Brose K, Jackson V, Hamada H, Pardoll D, Mulligan RC. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci USA. 903539-43, 1993

Isolation of BMDM from mouse femurs

Kathleen Kennedy 2005

Modified by committee 1/07

Remove femurs (Day 0)

• Use Female Mice age 8-12 weeks
• Soak extracted femur in 70% EtOH for no more then 1 minute.
• Remove excess tissue and the "knee" from femurs using a dry Kimwipe, or scalpel.
• Store femurs in RPMI on ice until BM is extracted. Goal is no more than 30 min from time mouse dies until BM is extracted Ð so do only as many mice at one time as you can to accomplish this.

Flush femurs with complete RPMI

• NOTE: all media/PBS used at every step in this protocol should be pre-warmed to 37°C.
• Media = 500ml of RPMI + 50ml of FBS, 5 ml Pen/Strep, 5ml L-glutamine. Media should be no more then 2 months old
• Add rh-MCSF to media just prior to use (final concentration 50ng/ml). rh-MCSF should NOT be freeze/thawed. Thaw only once and then store at 4 degrees. Do NOT store diluted M-CSF. OK to store undiluted rh-MCSF for up to 2 months.
• Fill a 10 mL syringe with ~ 8.5 mL RPMI and affix a 26 5/8 gauge needle.
• Snip off the head of the femur (knob that goes into hip).
• Drill the needle into the "crown" at the other end of the femur (where the knee was).
• Flush each femur with 8.5 ml RPMI into a 50 ml conical tube.
• Disperse marrow using a 10 ml pipet, then strain through a 70 um nylon cell strainer into another 50 ml conical tube. Rinse strainer with 5 ml of complete media.
• Count the cells. Use Turks at 1:1 to lyse the red cells. Count 4 corners.
• Plate 5x10⁶ cells/10 cm dish in 10mls of media on non-TC coated 10 cm Petri dishes.

On day 4 wash the cells Ð NOTE: The day of harvest is Day 0. Example: Harvest on a Friday (Friday is day 0). Wash on the following Tuesday (Day 4). Replate on Thursday (Day 6). Experiment on Friday. The day of the experiment is always the same day as the day of harvest.

• Wash - rock plate, aspirate media, add 7-10 mLs of warmed plain RPMI with (no additions), rock again, aspirate again.
• Add 10 mL complete RPMI with rh-MCSF to each dish.

On day 6 lift cells, count and replate on tissue culture-treated plasticware.

• Rock plate, Remove RPMI, wash x1 with 7-10mls PBS (warmed) add 7 mL PBS + 1 mM EDTA and incubate at 37°C for ~ 5 minutes.
• Lift cells off the bottom of the plate using a 10 mL pipet.
• Add cells from 4 dishes to a 50 mL conical containing 10 mL complete RPMI (now has 38mls in it).
• Remove the remaining cells from the 4 plates: Add 5mls of PBS/EDTA to plate 1, then transfer this to plate 2, then to plate 3, then to plate 4. Then add this 5mls to the 50ml conical tube. Repeat this process 1 more time (sequentially washing any remaining cells from each plate then adding to the conical).
• Collect cells by spinning at 1500 RPM for 5 minutes in centrifuge. Aspirate the supernatant.
• You will now have two 50ml conicals with cell pellets. These pellets need to be resuspended and combined. To do this, add 10mls of complete RPMI to the first tube, resuspend the pellet and transfer it to the second tube. Resuspend the pellet from the second tube. Rinse the 1^st tube by adding 10mls of complete RPMI to it and transferring to the second tube. Bring the final volume to 40mls by adding an additional 20mls of media.
• Count cells (dilute 1:1 with Trypan Blue and count at least 4 corners of the hemacytometer) and replate 1 million cells in 1.5mls of media in a well of a 6 well TC-treated plate in complete RPMI with rh-MCSF.
• Cells should be plated 18-24 hours prior to beginning the experiment.

Perform experiment on day 7.

• We remove the media and replace it with serum free RPMI (after washing the cells 3X with PBS).
• Cells are stimulated with known Toll agonists or infected with a pathogen.
• Media and plasma membranes are harvested at time points ranging from 4 hours to 24 hours.

Plate 1 X 10⁶ cells/ 6-well well.

Plate 1.552 X10⁷ cells per 150 cm² flask.

Concentration of Collected Supernatant

C. Lorang

2008

Equipment:

SW28 Rotor

Ultra Clear centrifuge tubes (beckman 344058)

Beckman Floor centrifuge

Amicon Ultra-15 Filter tubes (Millipore UFC900324)

Sterile PBS

Protocol:

• Thaw media. Transfer to centrifuge tubes and spin in SW28 rotor at 10,000xG (27,500 RPM) at 4C to spin down organelles.
• Transfer the supernatant to the filter tubes that have been previously buffered with sterile PBS.
• Spin the sample through at 1,500xG (2,500 RPM) at room temperature.
• Combine concentrated samples and spin down to a volume of ~ 500ul.
• Add 15ml of sterile PBS and filter to a similar volume.
• Freeze sample at -80C until needed.

Mass Spec Analysis of Serum Isolated from Mice Infected with Salmonella

Protocol:
Updated 8/20/09, S. Fallen, C. Lorang

Step 1: Grow Bacteria, Determine CFU, and Dilute to proper concentration

1. Inoculate 3ml of LB broth + kanamycin (1000x) with a single colony of salmonella from kan+ agar plate. Incubate O/N at 37°C with shaking.
2. Dilute culture 1:10 and determine OD. For Salmonella, expect OD between 4-5.5.

   1 OD = 1x10^9 cfu/ml.

3. Add X ul of culture to 1 ml final volume PBS to get 4 x 10^8 cfu/ml. Label tube #1.

   X ul = (4x10^8 cfu x 1000 ul/ml) / (___OD x 1x10^9 cfu/ml/OD). Expect to add 80-100ul of culture.

4. Set up 7 more eppendorf tubes and label #2-#8. To tubes 2-5, add 900ul PBS. To tubes 6-8, add 750ul PBS. Dilute the culture as follows:
   Add 100ul culture from tube #1 to #2, vortex.
   Add 100ul culture from tube #2 to #3, vortex.
   Add 100ul culture from tube #3 to #4, vortex.
   Add 100ul culture from tube #4 to #5, vortex.
   Add 250ul culture from tube #5 to #6, vortex.
   Add 250ul culture from tube #6 to #7, vortex.
   Add 250ul culture from tube #7 to #8, vortex.
   
5. Take 100ul of culture from tube #5 and add to 9.9ml of PBS. This is the working concentration of 4x10^2 salmonella/ml. Save for step 2.
6. From tubes #5-#8, spot 10ul 4 times onto a kan+ agar plate. Incubate plate at 37°C O/N. Spots from #5 should produce 400 colonies, #6 = 100 colonies, #7 = 25 colonies, and #8 = 6 colonies.

Step 2: Infect Mice IP with Salmonella

1. Take up working concentration of the Salmonella culture in a 1cc syringe and attach a 27G1/2 needle. Also prepare syringes with sterile PBS for injections into control mice.
2. Make intraperitoneal injections of 250ul of the salmonella (100CFU) into each of 12 mice. These are the infected mice.
3. Make intraperitoneal injections of 250ul of PBS into each of 4 mice. These are the control mice.
4. Monitor the health of the mice daily until they are euthanized.

Step 3: Euthanize mice and harvest serum, organs of interest.

1. On day 3, euthanize mice by carbon dioxide inhalation and cervical dislocation.
2. Using a 5ml syringe and a 22G1 needle, inject 3ml PBS into the peritoneal cavity of the mouse. Swish the fluid around by gently pressing on the stomach several times. Aspirate the intraperitoneal wash using the same syringe and transfer to 1.5ml eppendorf tubes in 1ml aliquots.
3. Using a 1cc syringe and a 27G1/2 needle, puncture the heart and aspirate as much serum as possible. Transfer to a serum separator tube and let sit at RT for 20 minutes. Spin down at max. speed for 5 minutes and transfer plasma (top layer) to PCR tubes in 50ul aliquots. Freeze immediately.
4. Remove the spleen and liver and place into wells of a 12-well plate containing 500ul PBS. Keep on ice until next step.

Step 4: Determine CFU in mice.

1. Weigh the harvested liver and spleen and place into safe-lock eppindorf tubes containing 500ul sterile PBS and a steel bead for homogenization. Homogenize tissue for 3min at 30 oscillations/sec and transfer to a fresh tube.
2. Remove 1ml of collected peritoneal wash and spin at full speed to pellet cells. Resuspend cells in 200ul of 1% Triton PBS.
3. Set up dilution plates A and B as follows:
   1. Aliquot 100ul of homogenized tissue or IP wash into row "a" of a 96 well plate.
   2. Aliquot 150ul of sterile PBS into row "c, e, g" of plate A and "a,c,e,g" of plate B.
   3. Do a 1:4 serial dilution by removing 50ul of sample and diluting into subsequent row (row c of plate A). Repeat for each serial dilution, changing tips between each dilution; i.e. remove 50ul from row c and add to row e of plate A. Repeat until all 8 rows have been diluted with sample.
4. Label LB plates containing Kanamycin antibiotic to reflect organ and dilution plate (LIVER dilution A). Spot 10ul of serial dilution in quadruplet on plate (as in diagram below). Do this for dilution plates A and B for the liver, spleen, and peritoneal wash.

   

5. Incubate overnight at 37°C.
6. The next morning, count the grown colonies in each spot.
7. Calculate the CFU/mg/ml by the following equation. This will give you the level of infection for each organ spot, average the 4 spots from each organ and plot on a graph to determine and compare infection status.
   (Colonies counted/ (Dilution factor x 10)) X (Volume of PBS in ul/ mass of tissue in mg)

Step 5: Run ELISA and RT-PCR analyses on tissue and plasma specimens to confirm activation.

ELISA:	Follow ELISA protocol attached below. Samples: Plasma diluted 1:20 Antibodies: IL12p40, IL6, IL10, IL1beta, TNFalpha, and any others of interest.
RT-PCR:	Follow the manufacturers' protocols for Trizol reagent, Turbo DNA-free, and SuperScript II RT for RNA isolation and cDNA synthesis.
	PCR mixtures: 1x EF1alpha (control) ABI primer probes 6ul primers 1ul primer/probe 2ul 2mM probe 7ul ddH20 10ul Taqman 10ul Taqman Fast 2ul cDNA template 2ul cDNA template Samples: Spleen and liver homogenates Primers: IL12, IL6, IL1beta, MP2, and any others of interest.

Step 6: Prepare plasma for mass spec (reduce, alkylate, digest, glycocapture)

Step 7: Run samples on spectrometer of choice.

Step 8: Analyze MS results.

ELISA

Day 1

Day 2

Reagents

1. 50ul human/mouse serum
2. Trifluoroethanol (TFE, Sigma)
3. Affi-Prep hydrazide resin, 50% in isopropanol (cat# 156-0016 Bio-Rad)
4. Oxidation Buffer : 20mM sodium acetate, 150mM sodium chloride, pH5.0
5. Coupling buffer : 100mM sodium acetate, 1M sodium chloride, pH4.5
6. Denaturing Buffer: 8M Urea, 0.5%SDS, 20mM Tris-HCl, pH 8.3
7. 1.5M sodium chloride
8. 80%ACN 0.1%TFA
9. 0.1%TFA
10. 1%TFA
11. MilliQ or equivalent dH2O
12. PNGase F (NEB or Prozyme Glyko N-Glycanase 2.5U/ml)
13. Desating colums (500mg tC18; 96-well uElution MCX plate, Waters)
14. Sequencing grade modified trypsin (Promega; V5113; 20ug/tube)
15. 0.7ml glass vials (Waters)
16. 15ml glass tube
17. 5mL tubes with caps "FACS tubes"
18. 2mL eppendorf
19. 0.65mL eppendorf

*Reagents to be made fresh

1. Sodium periodate: 100mM in H2O (21.4mg in 1mL H2O, light sensitive)
2. Tris(2-carboxyethyl) posphine: 200mM in water (TCEP, Pierce, Rockford IL, 57.33mg in 1mL H2O) 25x.
3. Iodoacetamide: 250mM in water(46.5mg/ml in water, light sensitive)
4. 100mM and 50mM Ammonium Bicarbonate pH 8.3
5. 10%NH4OH/90%MeOH solution fresh. Dilute NH4OH(~28-30%stock) 2mL in 10mL 100%MeOH. Then QS to 20mL with MeOH.

Procedure:

DAY 1
1. Dilute 50 µl of plasma with 50 µl 100mM NH4HCO3 solution (pH 8.3) to a final volume of 100 µl in 2mL eppendorpf tube, mix well;
2. Slowly add 100 µl of TFE to plasma, vortex for 30sec;
3. Add 8 µl of TCEP with 30 min incubation at RT.
4. Add 10 µl of Iodoacetamide with 30 min incubation at RT in the dark.
5. Add 1.7 ml of 100mM NH4HCO3 solution (pH 8.3), mix well before addition of trypsin 40ug.(1:100 trypsin vs protein)
6. Cover with parafilm and digest at 37°C for overnight with gentle shaking or waterbath (save ~20µg of peptides to check on SDS-PAGE for completion of tryptic digestion, load 20 µg of undigested plasma as control)
   DAY 2
7. Check trypsin digest on SDS-PAGE gel.
   Invitrogen NuPage gel Protocol Bis-Tris gel/Reduced sample protocol
   1. Prep and wash precast gel.
   2. Clamp gel into container and fill front and rear container with 600mL of MOPS buffer.
   3. Add 500uL of NuPage Antioxidant to 200mL MOPS buffer, mix, and add to the inner chamber.
   4. For 10uL reaction:
      1. 2.5uL Sample Buffer
      2. 1uL Reducing Agent
      3. XuL sample
      4. QS to 10uL with water
   5. Heat at 70c for 10min, Load on gel run at 200V for 50min
   6. Wash gel with dH2O for 45 sec in the microwave DO NOT ALLOW TO BOIL and rinse with new dH2O, repeat 3x.
   7. Add Simply Safe Stain to cover and microwave for 45 sec DO NOT ALLOW TO BOIL. Allow to incubate for 10min on rocker/mixing plate.
   8. Destain with dH2O. Tryspinized samples should be clean and have no bands.
   9. If bands are present add additional trypsin and allow to digest overnight. Recheck digestion the next day.
   SAMPLES CAN BE FROZEN AT THIS STEP

8. Adjust pH to ~3.0 before add sample to prepared C-18 desalting column. pH sample with 1%TFA (~1ml)
   tC-18 500mg Sep-Pak cartridge protocol/vacuum manifold
   (5-10mg protein, do not exceed vacuum pressure of -5 inHg.)
   1. Wet column with 5mL 100%MeOH
   2. Prep with 5mL 80%ACN,0.1%TFA buffer
   3. Equilibriate with 10mL 0.1%TFA
   4. Load sample into column make sure pH<3
   5. Collect and re-load flow-through in column
   6. Wash column with 10mL 0.1%TFA
   7. Elute sample in glass tube with 4mL 80%ACN/0.1%TFA.
   
   
9. Collect the eluted sample to a new glass tube and dry down in SpeedVac ~3hrs;
   SAMPLES CAN BE FROZEN AT THIS STEP

10. Re-suspend peptides in 450 µl oxidation buffer, vortex for at least 1min. Double check the solubilization of sample, sonicate if necessary. Add 50 µl of sodium periodate to each sample, and incubated in dark at RT for 1 hour.
11. While incubating sample, prepare 150 µl of pure Hydrazide resin (300 µl of 50% slurry) per sample by washing with 3X 1 ml of Milli Q water followed by washing/equilibration with 3X 1 ml of coupling buffer. Resuspend Hyrdazide resin in 300uL coupling buffer in 5mL FACS tube.
12. Adjust ph<3 of sample and clean up with C-18 desalting column (see cartridge protocol.) Elute with 2 ml of 80%ACN/0.1%TFA into tube with the prepared hydrazide resin. Cover with parafilm and conjugate the glycopeptides to hydrazide resin at RT with end over end mixing overnight.
    DAY 3
13. Remove and discard supernatant and transfer resin into 1.7ml eppendorf and spin down to remove excess supernatant.
14. Wash/Remove supernatant by spinning at 3000 X g for 2 minutes with 3x1 ml each of
    • Denaturing buffer
    • 100mM Ammonium Bicarbonate Buffer
    • 1.5M NaCl
    • 80% ACN
    • Ultra pure H2O
    • 50mM Ammonium Bicarbonate Buffer.
15. Transfer/Resuspend resin with 300uL 50mM Ammonium Bicarbonate buffer in 0.65mL eppendorf.
16. Add 1uL PNGase F and cover with parafilm and incubate at 37°C with mixing overnight.
    DAY 4
17. Spin down and save the supernatant to a new 1.7mL eppendorf and extract the hydrazide resin twice with 200 of 80% ACN/0.1%TFA. Spin down 3000g x 2min, avoid sucking up resin.
18. Transfer supernatant into 1 ml glass vial and dry down to less than 100 µl. Clean up sample with 96 well MCX plate.
    96-well MCX plate protocol vacuum manifold
    1. Wet filter with 800uL MeOH
    2. Wash filter with 800uL 0.1%TFA
    3. Bring sample to pH<3 with 1%TFA and load to column
    4. Collect flow-through in glass tube and re-run sample through column
    5. Wash column with 800uL 0.1%TFA
    6. Wash column with 500uL 80%ACN/0.1%TFA
    7. Wash column with 500ul H2O
    8. Elute sample with 500uL 10%NH4OH/90%MeOH(***make solution fresh***) into glass tubes
    
    
19. Elute the peptides from MCX column in the glass vial, and dry down in SpeedVac. (~30min)
20. Resuspended in 40 µl of 0.1% FA/1% ACN for mass spectrometry analysis (4.5 µl in mass spec tube). Store remaining sample at -20C.