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Products for this protocol

• Acetic acid
   
  
• Acetonitrile
   
  
• Derivatized silica resin (5 µm, C18)
   
  
• Ethanol (70%)
   
  
• Helium gas (pressurized)
   
  
• Methanol (50%)
   
  
• Protein sample of interest
   
  

• Autosampler (e.g., Dionex FAMOS)
   
  
• C18 trap cartridge (Michrome Bioresources)
   
  
• Capillary cutter (Scientific Instrument Services)
   
  
• Divert valve (six-port)
   
  
• Forceps
   
  
• Hex driver
   
  
• HPLC system (e.g., the Agilent 1100 HPLC)
   
  
• Mass spectrometer (e.g., Thermo Finnigan ITMS)
   
  
• Microcentrifuge tubes (1.7 ml)
   
  
• MicroCross with 0.006-inch through-holes (Upchurch Scientific)
   
  
• A microelectrospray ionization device available from Brechbuhler, Inc. is used to hold the MicroCross in place.
   
  
• Microscope (low-resolution)
   
  
• pH paper
   
  
• Polyimide-coated capillary (75 µm I.D. x 360 µm O.D.) (PolymicroTechnologies)
   
  
• Pressure cell (Brechbuhler, Inc.)
   
  
• Propane torch
   
  
• Regulator (two-stage, high-pressure)
   
  
• Sonicator
   
  
• Spatula
   
  
• Teflon ferrules (Chromatography Research Supplies)
   
  
• Tubes (15 ml polypropylene) (optional; see Step 15)
   
  
• Vortex mixer
   
  

1. Cut a 30-cm long piece of 75 µm I.D. x 360 µm O.D. polyimide-coated capillary.
    
   
2. Hold one end of the capillary with forceps. Remove 2-3 cm of polyimide by passing it through a flame from a propane torch.
    
   
3. Pull the capillary slowly to make a straight tapered tip.
    
   
4. Clean index finger by rinsing with 70% ethanol.
    
   
5. Place the tapered end of the capillary on the cleaned finger. Carefully trim the end with a capillary cutter. Rinse the tapered tip with 70% ethanol.
    
   
6. Inspect the tip under a microscope (5X-10X magnification) to ensure that the tapered tip is blunt (i.e., square and not jagged) and ~5 µm across.
    
   Alternatively, purchase the tips from New Objectives Inc. or purchase a laser puller from Sutter Instruments Inc.
    
   
7. Using a spatula, place ~50-100 µl (dry volume) of 5-µm C18-derivatized silica resin in a 1.7-ml microcentrifuge tube.
    
   There are many derivatized beads from which to choose. Particle size directly affects back pressure and chromatographic fidelity, but in general, 5-µm particles provide good results. Novices should considering using POROS (Appelera) beads, which are very easy to work with because they generate lower back pressure.
    
   
8. Add 500 µl of 70% ethanol to the C18 resin. Vortex. Sonicate briefly (i.e., no more than 1 min).
    
   The viscosity of the solvent can be modified to affect the packing rate. Due to the occurrence of apparently irreversible changes in the C18 beads that adversely affect separation, prepare the slurry daily.
    
   
9. Place the tube containing the slurry in the pressure cell.
    
   Handle the slurry-filled tube with forceps. As the chamber is drilled to accept tubes of various lengths, it may be necessary to place some Kimwipes in the bottom to bring the tube containing the slurry to a height inside the chamber that is easy to use.
    
   
10. Thread the blunt untapered end of the capillary into the top of the pressure cell (i.e., through the nut, the Teflon ferrule, and the top of the pressure cell). The end of the capillary inside the pressure cell should sit about halfway down into the 500 µl of slurry.
     
    Looking across the top of the pressure cell chamber, it is possible to observe when the capillary begins to bend, and thus determine that it is at the bottom of the vial.
     
    
11. Pull the capillary up about 0.5 cm. Close the top of the cell, making sure that the O-ring is in place. Tighten all four bolts with the hex driver. Secure the capillary in place by tightening the nut on top of the cell.
     
    Always secure all four bolts before turning on the helium gas. If a leak occurs, turn off the gas and reseat the O-ring or replace it with a new one and try again.
     
    
12. Set the two-stage, high-pressure regulator to 1000 psi. Slowly turn on the pressure in the cell by turning the valve 180°. Pack the capillary to 10 cm and stop, or pack the entire length of the capillary. Trim the length of the capillary column as desired.
     
    See Troubleshooting.
     
    
13. When the packing has come to within 0.5-1.0 cm of the desired length, turn off the pressure inside the cell.
     
    
14. Remove the slurry. Replace it with a vial of 0.1 M acetic acid.
     
    
15. Seal the cell as described in Step 11. Turn the pressure on and let the capillary rinse at least until the effluent turns acidic (check with pH paper).
     
    This wash will finish the packing, as some unpacked silica remains in the back of the capillary. The column can be stored indefinitely by placing the end inside a 15-ml polypropylene tube filled with 50% methanol and sealing the top.
     
    
Microsprayer Setup
 

16. Set up the automated µTrap-µLC-ESI-MS/MS system (Fig. 1 ).
     
    
     
    Figure 1. Diagram of an automated µTrap-µLC-ESI-MS/MS system. A tapered ESI emitter packed with C18 (Port 1), a high-voltage lead (Port 2), a miniature C18-packed peptide µtrap cartridge (Port 3), and a six-port divert valve (Port 4) were connected to a PEEK four-way union. Peptides were loaded for desalting on the C18 µTrap cartridge with the six-port divert valve open. When closed, the flow is redirected to the ESI emitter/C18 µLC column.
     
    
    1. Insert the blunt end of the packed capillary column into the MicroCross four-way union at Port 1.
        
       
    2. Place the tapered tip within 1-2 cm of the MS orifice.
        
       
    3. Attach the voltage supply to Port 2. Set voltage to ~2 kV.
        
       
    4. Attach the C18 trap cartridge to Port 3.
        
       
    5. Connect the six-port divert valve to Port 4.
        
       
17. Prior to sample analysis, perform a system suitability check using a well-characterized sample (preferably a purified peptide in the same class as that to be analyzed) to determine whether all of the components are functioning properly.
     
    
    1. Check the calibration of the mass spectrometer by comparing observed mass to theoretical mass (e.g., see Fig. 2a ).
        
       
    Figure 2. Example of multiple-charged peptide mass spectrum. The three most abundant charge states of neurotensin (a), the chromatographic total ion current trace at the point where neurotensin eluted showing an average peak width at half-height (c), and the same mass spectrum as in a, but with the ordinate axis expanded 200X normal to demonstrate how to check for signal/noise (b).
     
    See Troubleshooting.
     
    
    2. Compare measured to expected retention times.
        
       See Troubleshooting.
        
       
    3. Monitor back pressure at the HPLC.
        
       
    4. Calculate peak width of a single analyte by plotting the single ion current trace (Fig. 2c).
        
       See Troubleshooting.
        
       
    5. To assess the "cleanliness" of the solvents and sample, measure the signal-to-noise at a point where an analyte elutes by summing mass spectra across the point of elution. The ratio of signal to noise is estimated by expanding the ordinate axis to the point at which individual charge states are obscured by the noise (Fig. 2b).
        
       An analyte may "drag" along uncharacterized components by electrostatic and hydrophobic interactions that decrease the ratio of signal to noise. Aging solvents can increase the noise floor. See Troubleshooting.
        
       
    6. To ascertain whether the mass spectrometer is tuned properly, measure the ratio of signal to noise at the point when an analyte elutes.
        
       Because mass spectrometric data are not absolute, monitoring the signal-to-noise ratio will be more meaningful than signal response alone. See Troubleshooting.
        
       
18. Load the sample of interest on the trap cartridge precolumn.
     
    
19. Wash the column with the six-port divert valve open so that flow enters the four-way union at Port 3 and exits at Port 4.
     
    
20. After washing, close the divert valve so the flow is redirected to the C18 capillary column in Port 1.
     
    This type of setup using a trap cartridge provides extended life for the separation column. The device shown in Figure 1 can be used without the trap cartridge in Port 3 if desired. Keep the six-port divert value closed if not using the trap.
     
    
21. Elute the column with a fast linear gradient (e.g., 0%-60% acetonitrile over 15 min).
     
    Gradient conditions may vary depending on the peptide under analysis. Flow rate through the column will also vary depending on internal diameter and packed length. For a 75-µm capillary column packed to 7 cm, the flow rate can be as low as 150 nl/min when optimized. To compensate for the decrease in flow that occurs with a decrease in viscosity (i.e., higher-percentage acetonitrile has lower viscosity relative to H2O) when using a restrictive flow splitter of constant length, an increasing flow rate can be programmed at the HPLC pump over the duration of the linear acetonitrile gradient.
     
    

• Step 12
  • Problem: The silica beads do not appear to flow during column packing.
     
    Solution: Try one or all of the following:
     
    
    • Turn the pressure inside the cell on and off several times to dislodge any air pockets in the slurry.
       
      
    • Vortex the slurry again; the slurry may have settled to the bottom of the tube. Alternatively, add a micro-stir bar and perform the packing on a magnetic stir plate to keep the slurry suspended. Note that use of the stir bar can fragment the C18 beads, so it is best to prepare the slurry fresh daily.
       
      
    • Cut off a 0.5-cm length of capillary inside the cell and reposition; it may have become clogged.
       
      
    • Hold the capillary taut between the top of the pressure cell and the microscope stage and vigorously flick the capillary so that it vibrates like a violin string.
       
      
• Step 17
  • Problem: The calculated and observed molecular weight for the analyte does not match within the tolerance of the mass spectrometer.
     
    Solution: Clean and tune the mass spectrometer.
     
    
  • Problem: Elution time for the calibration peptide is outside of the expected range.
     
    Solution: Check the HPLC system to ensure it is functioning as programmed, or replace the microcolumn.
     
    
  • Problem: The peak shape for the calibration peptide is not symmetrical or is too broad (for a 0.5-1.0-pmol injection of peptide standard, it should be ~10-30 s) (Fig. 2).
     
    Solution: There is likely a problem with the packing material, or dead volume has been introduced somewhere in the µLC column. Replace the column with a freshly packed one.
     
    
  • Problem: The signal-to-noise ratio of the analyte is below an acceptable value.
     
    Solution: Change the solvents. Retune the mass spectrometer. Clean the lenses involved in focusing ions. When a lens becomes coated with even a thin invisible film (e.g., from repeated analysis of samples), then the applied voltage is no longer equal to the field voltage experienced by the ions during transmission, and a decrease in performance (e.g., signal to noise) is observed.