This protocol offers step-by-step instructions for preparation of raw blood plasma for liquid chromatography – tandem mass spectrometry (LC-MS/MS) analysis in clinical proteomics studies. The entire transformation from plasma proteins to desalted tryptic peptides takes only 3–4 h. The protocol can be adopted for large-scale studies and automation.


We describe a protocol for Lipidomic analysis of tissue culture cells, tissues, and purified organelles. Lipids are a class of molecules that have roles in energy storage, plasma membrane integrity, and signaling events. This protocol provides direction on how to extract lipids from plasma, cells, tissue, and purified organelles for analysis by liquid chromatography (LC)-MS.


We describe a protocol for multiplexed proteomic analysis using neutron-encoded (NeuCode) stable isotope labeling of amino acids in cells (SILAC) or mice (SILAM). This method currently enables simultaneous comparison of up to nine treatment and control proteomes. Isotopologs of lysine are introduced into cells or mammals, via the culture medium or diet, respectively, to metabolically label the proteome. Labeling time is ∼2 weeks for cultured cells and 3-4 weeks for mammals. he resultant peptides are chromatographically separated and then mass analyzed. During mass spectrometry (MS) data acquisition, high-resolution MS1 spectra (≥240,000 resolving power at m/z = 400) reveal the embedded isotopic signatures, enabling relative quantification, while tandem mass spectra, collected at lower resolutions, provide peptide identities. Both types of spectra are processed using NeuCode-enabled MaxQuant software. In total, the approximate completion time for the protocol is 3-5 weeks.


Isotopic labeling is commonly applied for investigating intracellular metabolism. The general workflow is to first introduce isotopically-labeled metabolites into the culture medium, then at defined time points wash and harvest cells, process samples for metabolomics analysis, and analyze the samples for isotopic enrichment in specified metabolite pools. Here we apply this technique to primary hepatocytes from mice. We introduce either 13C5 glutamine or 13C6 glucose at the typical media concentrations, 1:1 replacing the 12C version with 13C version. Cells are harvested at 0 and 30 min after isotope introduction, metabolites are extracted and then analyzed by GC-MS and LC-MS. The resulting data are used to compare relative 13C isotopic labeling in metabolites between different genetic mutants. This strategy is not suitable for directly quantifying metabolic flux (i.e., Metabolic flux analysis), but is useful for describing relative metabolic flux between two models. The expected time to complete is ~3-5 days.

  1. Resuspend tissue in 6M GnHCl 100 mM tris pH 8
  2. Probe sonicate until fully homogenized (excess material can be frozen here)
  3. QS with MeOH to 90% [MeOH]
  4. Centrifuge 9,000x g for 5 min
  5. Decant (remove excess supernatant with pipette)
  6. Air dry 5 min
  7. Resuspend in 8M urea & 100 mM Tris (should be <3 mg/ml)Probe sonicate until resuspended
    • Probe sonicate until resuspended
  8. Quantify protein by BCA assay (or method of choice)
  9. Add 10 mM TCEP, 40 mM chloroacetamide
  10. Add LysC 50:1, incubate 4 hrs
  11. QS with 100 mM tris to 2 M urea
  12. Add trypsin 50:1, incubate overnight
  13. Add TFA to pH ~1
  14. Centrifuge 9,000x g for 5 min

 

Desalting (≤300 µg)

10 mg Strata X, 1 mL volume (reversed phase solid phase extraction cartridge)

Note: Use gravity or a vacuum manifold

  1. 1 mL 100% ACN
  2. Fill cartridge with 0.2% FA
  3. Load sample
  4. Fill cartridge with 0.2 % FA
  5. Elute into clean tube with 300 µl 80% ACN 0.2% TFA.
  6. Dry down reconstitute in 0.2% FA.

 

Desalting (≤1 mg)

30 mg Strata X, 3 mL volume (reversed phase solid phase extraction cartridge)

Note: Use gravity or a vacuum manifold

  1. 3 mL 100% ACN
  2. Fill cartridge with 0.2% FA
  3. Load sample
  4. Fill cartridge with 0.2 % FA
  5. Elute into clean tube with 900 µl 80% ACN 0.2% TFA.
  6. Dry down reconstitute in 0.2% FA.

 

Desalting (≤5 mg)

60 mg Strata X, 3 mL volume (reversed phase solid phase extraction cartridge)

Note: Use gravity or a vacuum manifold

  1. 3 mL 100% ACN
  2. Fill cartridge with 0.2% FA
  3. Load sample
  4. Fill cartridge with 0.2 % FA
  5. Elute into clean tube with 1.5 mL 80% ACN 0.2% TFA.
  6. Dry down reconstitute in 0.2% FA.

 

Desalting (≤10 mg)

100 mg Strata X, 3 mL volume (reversed phase solid phase extraction cartridge)

Note: Use gravity or a vacuum manifold

  1. 3 mL 100% ACN
  2. Fill cartridge with 0.2% FA
  3. Load sample
  4. Fill cartridge with 0.2 % FA
  5. Elute into tube with 2 ml 80% ACN 0.2% TFA.
  6. Dry down reconstitute in 0.2% FA.

 

Peptide concentration

Resuspend sample in 0.2% FA, targeting ~2 µg/µL

Use Peptide colorimetric assay for peptide quantitation.

 

  1. Resuspend pellet in 6M GnHCl 100 mM tris pH 8
  2. Boil 5 min
  3. Rest 5 min
  4. Boil 5 min
  5. QS with MeOH to 90% [MeOH]
  6. Centrifuge 9,000x g for 5 min
  7. Decant (remove excess supernatant with pipette)
  8. Air dry 5 min
  9. Resuspend in 8M urea & 100 mM Tris (should be <3 mg/ml)
    • Probe sonicate until resuspended
  10. Quantify protein by BCA assay (or method of choice)
  11. Add 10 mM TCEP, 40 mM chloroacetamide
  12. Add LysC 50:1, incubate 4 hrs
  13. QS with 100 mM tris to 2 M urea
  14. Add trypsin 50:1, incubate overnight
  15. Add TFA to pH ~1
  16. Centrifuge 9,000x g for 5 min

 

Desalting (≤300 µg)

10 mg Strata X, 1 mL volume (reversed phase solid phase extraction cartridge)

Note: Use gravity or a vacuum manifold

  1. 1 mL 100% ACN
  2. Fill cartridge with 0.2% FA
  3. Load sample
  4. Fill cartridge with 0.2 % FA
  5. Elute into clean tube with 300 µl 80% ACN 0.2% TFA.
  6. Dry down reconstitute in 0.2% FA.

 

Desalting (≤1 mg)

30 mg Strata X, 3 mL volume (reversed phase solid phase extraction cartridge)

Note: Use gravity or a vacuum manifold

  1. 3 mL 100% ACN
  2. Fill cartridge with 0.2% FA
  3. Load sample
  4. Fill cartridge with 0.2 % FA
  5. Elute into clean tube with 900 µl 80% ACN 0.2% TFA.
  6. Dry down reconstitute in 0.2% FA.

 

Desalting (≤5 mg)

60 mg Strata X, 3 mL volume (reversed phase solid phase extraction cartridge)

Note: Use gravity or a vacuum manifold

  1. 3 mL 100% ACN
  2. Fill cartridge with 0.2% FA
  3. Load sample
  4. Fill cartridge with 0.2 % FA
  5. Elute into clean tube with 1.5 mL 80% ACN 0.2% TFA.
  6. Dry down reconstitute in 0.2% FA.

 

Desalting (≤10 mg)

100 mg Strata X, 3 mL volume (reversed phase solid phase extraction cartridge)

Note: Use gravity or a vacuum manifold

  1. 3 mL 100% ACN
  2. Fill cartridge with 0.2% FA
  3. Load sample
  4. Fill cartridge with 0.2 % FA
  5. Elute into tube with 2 ml 80% ACN 0.2% TFA.
  6. Dry down reconstitute in 0.2% FA.

 

Peptide concentration

Resuspend sample in 0.2% FA, targeting ~2 µg/µL

Use Peptide colorimetric assay for peptide quantitation.

Note: Requires a pressure bomb attached to a tank of compressed helium

  1. Analytical columns with integrated emitter tips are manufactured using 360-µm o.d. × 75 µm i.d. fused silica capillary tubing.
  2. To form the emitter tip, use a butane lighter to remove ~3 cm of polyimide coating 5 cm from one end of the fused silica.
  3. Clean this area with methanol to remove any remaining charred polyimide coating before inserting the silica into the puller.
  4. Using the manufacturer’s recommended settings for the laser puller, pull a 15-µm tip, by avoiding the polyimide coating.
  5. Inspect the tip under a microscope; if the tip is closed, etch with HF to create an opening. In the fume hood, transfer 50 µl of HF to a microcentrifuge tube and fill another tube with 0.5 M ammonium formate. Dip the electrospray tip in HF for 1–2 min. Then thoroughly rinse the tip with water.

! CAUTION Take appropriate safety precautions when using HF. Wear proper protective equipment, and perform the work in a properly vented fume hood. Wear safety goggles when you are working with the pressure bombs.

  1. Prepare a slurry of 5 µm BEH packing material by transferring the packing material to a glass vial containing a mini magnetic stir bar. Add ACN to the vial. As the 3.5-µm BEH packing material is added only to keep the smaller 1.7-µm material in the column, a very dilute slurry is recommended (0.1 mg, 3.5-µm packing material, 1 ml ACN). Place this into the pressure bomb.
  2. Insert the analytical column, with pulled tip pointing upward, through the high-pressure bomb. Be careful when you are handling the column, as the tip is fragile and easily damaged. Slowly adjust the position of the column until is 1–2 mm above the bottom of the tube.
  3. Set the pressure regulator on the helium tank to 1,000 psi.
  4. Increase the pressure in the pressure bomb by slowly turning the valve to the open position.
  5. Increase the pressure in the pressure bomb by slowly turning the valve to the open position.
  6. Fill ~5 mm of the tip with 5-µm BEH slurry. If desired, a lower pressure of helium (200–300 psi) can be used for this step.
  7. Prepare a slurry of 1.7-µm BEH packing material by transferring the packing material (~0.5 mg) to a glass vial containing a mini magnetic stir bar. Add 1 ml of chloroform to the vial.
  8. Fill the analytical column with 1.7-µm BEH packing material to a length of ~30 cm.
  9. Release the pressure by turning the valve to the closed position slowly to avoid column unpacking.

Reagents and materials

  1. 80% ACN with 6% TFA (!!make fresh everyday!!)
  2. 80% ACN
  3. 80% ACN with 0.5M glycolic acid (or lactic acid)
  4. 50% ACN with 1% ammonium hydroxide (make fresh)
  5. Ti-IMAC beads (MR-TIM005from ReSyn Biosciences)
  6. magnet

Note: Use 50 to 100 µL of Ti_IMAC beads for every 1 mg of peptide. 50 µL will be cleaner, but 100 µL will have higher phosphopeptide yield.

 

Procedure

  1. Wash beads three times with 1 mL 80% ACN with 6% TFA
  2. Resuspend sample in 1 mL 80% ACN with 6% TFA and combine with beads
  3. Vortex 20 minutes at ambient temperature
  4. Wash beads three times with 1 mL 80% ACN with 6% TFA
  5. Wash beads once with 1 mL 80% ACN
  6. Wash with 1 mL 80% ACN with 0.5 M glycolic acid
  7. Wash beads one times with 1 mL 80% ACN
  8. Elute two times with ~300 µL 50% ACN with 1% ammonium hydroxide
  9. Dry and desalt

 

Global Settings
  • Use Ion Source Settings from Tune = False
  • Method Duration (min)= 120
  • Ion Source Type = NSI
  • Spray Voltage: Positive Ion (V) = 2000
  • Spray Voltage: Negative Ion (V) = 600
  • Sweep Gas (Arb) = 0
  • Ion Transfer Tube Temp (°C) = 275
  • APPI Lamp = Not in use
  • Pressure Mode = Standard
  • Default Charge State = 1
Experiment 1
  • Start Time (min) = 0
  • End Time (min) = 120
  • Cycle Time (sec) = 2

Scan MasterScan

  • MSn Level = 1
  • Use Wide Quad Isolation = True
  • Detector Type = Orbitrap
  • Orbitrap Resolution = 60K
  • Mass Range = Normal
  • Scan Range (m/z) = 300-1350
  • Maximum Injection Time (ms) = 50
  • AGC Target = 2000000
  • Microscans = 1
  • RF Lens (%) = 30
  • Use ETD Internal Calibration = False
  • DataType = Profile
  • Polarity = Positive
  • Source Fragmentation = False
  • Scan Description =

Filter MIPS

  • MIPS Mode = Peptide

Filter ChargeState

  • Include undetermined charge states = False
  • Include charge state(s) = 2-6
  • Include charge states 25 and higher = False

Filter Dynamic Exclusion

  • Exclude after n times = 1
  • Exclusion duration (s) = 5
  • Mass Tolerance = ppm
  • Mass tolerance low = 10
  • Mass tolerance high = 10
  • Exclude isotopes = True
  • Perform dependent scan on single charge state per precursor only = False

Data Dependent Properties

  • Data Dependent Mode= Cycle Time
Scan Event 1
  • Scan ddMSnScan
  • MSn Level = 2
  • Isolation Mode = Quadrupole
  • Isolation Window = 0.7
  • Use Isolation m/z Offset = False
  • Multi-notch Isolation = False
  • Scan Range Mode = Define m/z range
  • Scan Priority= 1
  • ActivationType = HCD
  • Is Stepped Collision Energy On = False
  • Stepped Collision Energy (%) = 5
  • Collision Energy (%) = 30
  • Is EThcD Active = False
  • Detector Type = IonTrap
  • Ion Trap Scan Rate = Turbo
  • Scan Range (m/z) = 200-1200
  • Maximum Injection Time (ms) = 15
  • AGC Target = 40000
  • Inject ions for all available parallelizable time = False
  • Microscans = 1
  • Use ETD Internal Calibration = False
  • DataType = Centroid
  • Polarity = Positive
  • Source Fragmentation = False
  • Scan Description =