Janet B. Matsen:Baker Lab LCMS

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Revision as of 13:30, 20 June 2012 by Janet B. Matsen (talk | contribs) (PHYSICAL SETUP)

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Accela HPLC Protocol {Amanda got this from someone else}



  • Waters Acquity C18 Column
  • Also tried,
    • Hypersil Gold AX, 100 x 2.1 mm, Thermo-scientific
    • Hypersil Gold aQ, 100 x 2.1 mm, Thermo-scientific
    • PFP column: Hypersil GOLD PFP, 100 x 2.1 mm, Thermo-scientific
    • Hypersil GOLD 100 x 2.1 Thermo Scientific #25002-102130

Opening a New Column:

  • Record on spec sheet:
    • Date opened
    • Pressure and associated flow rate
    • Solvent used
  • Rinse all parts
  • Insert guard column cartridge into guard column
    • Don’t touch guard column cartridges while inserting it!
    • Use socket wrenches to tighten screw into bottom of the guard column.
    • Finger tighten guard column into the bottom of the real/separation column.
    • Cap both ends with screw-in end-caps.
    • Column is ready to be used.


    • A: Acidified H2O (H2O with 0.1% Formic Acid)
    • B: Acetonitrile 0.1% Formic Acid
    • C: Rotating Tap (Has our buffer on it now)
    • D: 100% Acetonitrile or Methanol or Isopropanol

Normal Wash:

    • 20% H2O
    • 80% Acetonitrile
    • 0.1% Formic Acid
    • 20 mM NH4Acetate

Our Wash:

    • Same as mobile phase C


Check these before running, you may not need to change any, or may need to adjust everything.

  1. Open an existing file (Janet started own folder on 6/20 from an old formate run of Amanda's)
    1. Study name: keep this general. Workbook: get more specific (date, etc.). You can have multiple workbooks per study.

Changing the Column

Flush previous column with their mobile phase D (whatever it is) before removing (100% acetonitrile, methanol or isopropanol, 0.5 mL/min, 10+ min)

    1. Open Xcalibur.
    2. Go to Instrument Setup --> Acela Pump (icon on left bar) --> Direct Control (words on top bar).
    3. Check the box next to “Take pump under direct control.”
    4. Change flow rate to 500 uL/min
    5. Type 100% next to solvent D (100% Acetonitrile)
    6. Press Play.
    7. Run for at least 10 minutes (a timer counts)
  1. Remove previous column
    1. Note: All necessary wrenches are inside the bottom of the autosampler box (AS). The pumps can continue while the column is being changed or they can be paused.
    2. Manually unscrew the output (top) of column
    3. (while holding column) Using two appropriate wrenches unscrew the very bottom connector from the metal HPLC input into the bottom of the guard column (all columns have a short extra column on the input with a small, 1cm “guard column” which is easily replaced if damaged. It is housed in a short metal pre-column).
    4. Place caps on the removed column and place back in the appropriate box, usually on top of the MS unit. (Box and end-caps for column in use should be stored on the top of the MS)
    5. Uncap the new column. (6/20/2012: acquity (blue box) pore size = 1.8 um, dimensions: 2.1x150 mm)
    6. Read direction of flow. Flow goes from the bottom up, so make the arrows on the column point up
    7. Attach bottom of new column, being careful to align threads to avoid leakage, using wrenches.
    8. Manually attach top plastic connector. Push red tubing in hole first, then screw in connector
    9. Dry connections so any liquid will indicate a leak
  2. Equilibrate column to starting conditions (& make sure column is on tight)
    1. In Acela Pump Direct Control, set starting conditions to run (e.g. 100% Solvent C, 0.4 mL/min).
    2. In Xcalibur, click instrument setup -- > “accela pump” menu  Direct Control. A new window opens. Check “take pump under control,” change parameters, then hit “Play”.)
    3. Pressure should equilibrate over ~1 min to ~570 bar. Check for leaks at the column and guard column, adjust as necessary.
      1. Adjust as necessary = lower flow rate if pressure > 700 ish. 6/20/2012: Amanda set the oven temp to 40oC because the pressure was too high.
    4. Run the column for 10 minutes to equilibrate and catch leaks. (10 min starts once you have 0.4 mL/min and pressure < 600 bar)

MS ionization unit: APCI (chrome)/ESI (matte brown) – Careful because parts may be hot! (we use brown)

  1. APCI is kept in a white box on the shelf above the computer, labeled APCI. The ESI is usually stored on top of the MS unit facing aack.
  2. Press button on front of MS to switch flow to waste
  3. Loosen the knob holding the ionizer head in the chamber and gently pull out the head, twisting to remove the alignment pin. Set aside head.
  4. Remove all cables from the ionization chamber, open up the two fasteners and pull directly back to remove (don’t forget the waste hose!)
  5. To put in ESI, remove the pin inside the chamber with needle nose pliers, rinse with methanol, place pin inside Ziploc bag in apci box.
  6. To put in APCI, take the pin from the Ziploc bag in apci box, rinse with methanol, dry with kimwipe, and place into pin-holder in the chamber. Touch the pin with neither bare nor gloved hands, lest you besmirch it.
  7. wipe down cone that points out at you if dirty (there is some rust/crust)
  8. Place chamber back on MS unit, closing fasteners
  9. Insert new head, lower to appropriate depth (C in my protocols), tighten
  10. Attach all cables. Note that for APCI the high voltage connects to the chamber directly, not to the back of the ionizer. Also note that for ESI the sample input line from the valve goes first through a metal connector (to ground) and a separate input line connects that metal intermediary on the chamber to the back of the ionizer.

Change mobile phases, if necessary

  1. Remove filter line from old bottle
  2. Rinse filter line with the new mobile phase
  3. Put filter line in bottle of new buffer.
  4. Attach a syringe to the line coming out of the purge valve in the pump
  5. Open the purge valve
  6. Start purging the line
    1. Open Xcalibur.
    2. Go to Instrument Setup --> Acela Pump (icon on left bar) --> Direct Control (words on top bar).
    3. Check the box next to “Take pump under direct control.”
    4. Change flow rate to 1000 uL/min
    5. Type 100% next to the mobile phase you’re changing
    6. Press Play.
    7. Run for 20 min.
  7. Close purge valve.
  8. Remove syringe and dispose of content

Changing the Wash/Flush Buffer

  1. Remove filter line from old bottle
  2. Rinse filter line with the new flush (Amanda rinsed with water that was stored up there; can also rinse with water in a beaker. here "new flush" = water because our solvent is mostly water)
  3. Put filter line in bottle of new buffer
  4. Put paper towel in Autosampler where wrenches live to catch extra fluid.
  5. Wash Needle to flush lines
  6. Open Xcalibur.
  7. Go to Instrument Setup --> Acela AS (icon on left bar) --> Direct Control (words on top bar).
  8. Choose “Wash Needle” from the drop down box.
  9. Change volume to 6000 uL (6 mL)
  10. Make sure the source (reservoir) is set to “Flush Bottle”
  11. Press apply.
  12. Repeat 2 more times. (it is fast(1-2 minutes); you can hear a hum pulsing when it is going.)

Choose tray type

  1. Quit Xcalibur and open the Instrument Configuration program on the desktop
  2. Choose Acela Autosamper (AS) under Configured devices
  3. Choose the and the correct type (e.g. for plates, use 96 well microwell plate + carrier + riser and for vials, use 1.8 mL vials)
  4. Quit configuration program
  5. In Xcalibur under sequence setup (lower image) choose “Change  tray names” and choose new tray type. E.g. for plates choose 96 well microwell plate + carrier + riser
  6. Put/remove in carrier and riser as needed. For plates positions are enumerated A:A1, etc. (first A indicates first of three plates), while for tubes numbering begins A1, etc.

Preparing Samples


  1. Set up a method for a new run (usually already completed previously)
    1. Open Xcalibur and choose instrument setup
    2. Choose “Acela pump” to setup gradient, e.g. starting at 95% A to 5% A. Click in the last row and start typing to add a new section. After changing the gradient program, note how long it is and update the run length for the TSQ Quantum and Acela PDA.
    3. Choose TSQ Quantum to setup MS. Click the “…” button to select your tune (tsq) file and be sure to update the parameters from the relevant tune, e.g. switch to SIM if necessary, etc.
      1. Can add masses to be detected for each scan event by adding the parameters to the table at the bottom of the window.
  2. If you didn’t do so under Column directions, equilibrate column to starting conditions, ~10 minutes.
    1. Manually run at x% A. E.g. if the run starts at 40% A, run at 40% A, following instructions above under “(1) Column”
  3. Set up sequence.
    1. In windows, create a new folder for your run, usually in C:\Xcalibur\Data
    2. click “sequence setup”
    3. If you’re using a version of an old sequence, simply open, save as, and edit.
    4. Adjust “File Name” (e.g. compound1), Path (click to select), injection volume. Position goes A:A1… for plates, A1… for tubes. Click to browse for the desire method, already created previously.
    5. Use the “fill down” button (two downward pointing arrows) to copy values down or automatically increment plate values (A1, A2…) after selecting desired column.
    6. Usually a sequence should start and end with a blank, e.g. PBS
    7. Run stds at the beginning, beginning with lower concentrations first.
  4. Run the sequence by pressing the run sequence button (right pointing arrow with a sheet on top).
    1. To start just one or a set of the sequences select the relevant row and click run sample (right pointing arrow with a test tube)
    2. Before starting, make sure that all instruments needed are “on” in the Status tab. E.g. if mass spec is used in the method the machine should be “ready to download”
    3. In the window that opens, check/uncheck post-processing as needed, make sure that the Acella AS is the starting instrument, and make sure box is checked such that pumps turn off after runs.
    4. Click the “acquisition queue” tab to see the loaded set of sequences. If you need to stop any sequence, check the box and press the “delete” key.
    5. If not using the divert valve, make sure that the MS is set to load/detect.

PROTEIN SETUP (EXAMPLE) – I don’t use this {Amanda}

Overall guidelines: - Generally use 1mg/ml protein, absolute max is 2 mg/ml; at higher concentrations precipitation will clog the switches - 10-15 ul sample per vial, with insert - 50 ul sample per well in plates

Column: BioBasic-4, labeled “Current” Ionization: ESI head Buffers: Water/Acetonitrile each with 0.1% formic, equilibrate to 95% water/5% AceN Flow Rate: 500 uL/min (in rare circumstances use slower flow for higher sensitivity)

Setup template (open in xcalibur; saved in read-only format on Desktop): Protein_Analysis_Sequence_Template.sld Save the template under a different name/directory Adjust sample names; include a blank (e.g. PBS) in first and last run At run time, be sure that Processing boxes are checked for Qual and Programs.

ProMass will run automatically Mass error is +- 0.02% To view results: Promass results folder, index.html See data sheet on top of computer for common protein modification masses

To reprocess data, if anything goes wrong, hit the “summation” icon in xcalibur.


MS Tuning notes for Accela HPLC/MS:

Note: When not running, inject/waste mode, otherwise load/detect mode.

  1. Sample injection:
      1. Use direct injection of salt and buffer free sample solution. For example using 1mM of the molecule of interest in 50% AceN/50% 0.1% formic acid water. Do not use any buffer, e.g. HEPES, or include any salts, as they may build up in the MS.
    1. Start with direct injection (we didn’t do this):
      1. To directly inject use a single tube with the needle adapter (on top of the MS; usually the needle adapter would be attached to the T-junction).
      2. Rinse the syringe (250 uL or 500 uL as needed) with solvent (5-6 times)
      3. Load sample as prepped above. Syringe should be placed into the syringe injector after it is attached to the input tube. Follow instructions in step (2) to turn on syringe pump.
    2. After you see compound in direct injection, go to inline injection (we started with inline injection):
      1. Use the T-junction. Attach the input line from the switch to one leg of T, attach input from syringe to other leg, and attach third leg to input of ionizer. Using direct control of the pump (icon in Quantum tune with a syringe flowing in to three collector flasks) run the normal HPLC buffers – note that if the syringe is not loaded in the injector the pressure will push the syringe plunger out!
  2. Vanilla Tuning
      1. Open Quantum Tune and run the MS (click on “pause” button in upper-left corner so it becomes a green triangle)
      2. Before changing anything chose “save as…” to select a new name for your tune, so as not to save over someone else’s hard-won tune file!
    1. Choose MS only optimization
    2. Choose – or + at the big blue “-“ button, here the example shows negative optimization – thus the ions will be negative, with -1 mass. Here the molecule has a mass of 144.2 , so the optimization mass will then be 143.2.

Set the optimization mass based on the -/+ setting

    1. Start the pump – Go to Setup menu and choose “syringe pump settings”. In the window that opens turn pump to “on” and set a flow rate, e.g. 5 or 10 uL/min. In that window also set the syringe volume, e.g. 500 uL, as appropriate.
    2. Set up MS device parameters -- Press the “tuning fork/green flask” icon towards the middle (Optimize compound dependent devices) to adjust scan parameters and pick which ones will be optimized. The define scan section will open in the upper right corner (as shown in the figure). Click in the check boxes to the left of each parameter to allow the automatic optimization to adjust that value – e.g. above the tube lens offset will be optimized.
    3. Define the scan – press the “multicolored bubbles” (Define Scan) button. For the simplest case choose Full Scan in centroid mode (the vertical bars button), and do a scan from 50 to 500 Daltons.
    4. Start the optimization – Press start in the upper left section. The program takes over, automatically optimizing the given parameters to maximize signal – this is shown in the lower-right hand corner. When it is done it will offer to save optimization-scanning info as a pdf (save it or cancel). If all has gone well, the spectrum in the lower-left corner now shows a big peak for your compound. Save the tune file (*.tsq) and you’re done!
    5. After doing some tuning with direct injection be sure to switch to inline injection and optimize parameters there – take special note of the gas flow rates, which need to be higher for the inline injection (because the liquid flow rate is higher).
    6. Stop the MS running by clicking the green triangle in upper left hand corner. Quit the Quantum Tune Program.
  1. Non-Vanilla Tuning
    1. Hand optimization – if the tuning is done and is showing you a peak, but you want it to be stronger, turn on the optimize compound dependent devices window. Start the trajectory view (icon with a watch band and a big green peak) to show total current (TIC) as a function of time to allow you to keep track of whether signal is improving. Now adjust each parameter, e.g. capillary temperature by clicking on it and editing in the space below; some parameters may adjust slowly, such as temperature, so keep track of the “Readback” value. The photocopies over the monitor give guidelines for these parameters – e.g. 400 to 600 for Vaporizer temperature in APCI, etc.
      1. Change the scan type – press the Define Scan (bubbles) button. Choose SIM to focus on one region of mass rather than recording a full spectrum. Set the center and the width of that mass region, e.g. 142.9 and 0.01 to define a very narrow band around a mass. This will increase sensitivity by integrating for a much longer time at each mass.
      2. Change the MS mode – choose MS/MS in the Optimization Mode region. Check “adjust parent mass” and “step collision energy” boxes. Then click start to start optimization. First a normal ms optimization will be done (and the parent mass adjusted to the best peak). Then the collision energy will be adjusted until a maximum is found – the data are shown in the lower right hand corner. Expect a gradual peak as a function of collision energy with a well-defined maximum. If the optimization fails, however, there is no peak as a function of collision energy and the program reports a failure to find fragments. The compound is rock-solid and won’t be amenable to MS/MS.