MSF Standard Operating Procudures


This page lists the standard operating procedures employed in the IU-MSF. If there are no sample handling or analysis instructions on your sample report, these procedures were employed. Further information can be obtained by speaking with Jon or Angie. Click on one of the experiment types below for the procedure in question.

    Hi-Res CI     Hi-Res ESI     MALDI-TOF     Low-Res ESI     Bio Low-Res ESI     Low-Res GCMS  

High Resolution Chemical Ionization

Samples are typically diluted with several drops of dichloromethane. All mass spectra are recorded on a MAT-95XP Trap double focusing mass spectrometer . The instrument is calibrated with perflourokerosene (PFK) each day and PFK is introduced for internal calibration of each sample mass spectrum. Electron Ionization (EI) is performed with 70 eV between the filament and the ion volume. Positive chemical ionization (CI) is achieved by pressurizing the ion volume in the source to approximately 1 torr with ultra-high purity methane gas. The data are recorded and the mass axis internally calibrated using the CMASS module of the Xcalibur 1.3 software package. A representative mass spectrum is taken from the data file and formula matching is facilitated by the LIST module XCalibur. The precise experimental details depend on the molar mass of the analyte:

  • MW < 300 Da: 2 µL of the solution are injected into a Trace GC with a 0.25 mm i.d., 30 m long Restek RTX-5Sil-MS column running the following temperature gradient: 80 °C for 0.5 minutes, 15 °C/min to 250 °C, hold for 5 min @ 250 °C. The injector port and transfer line are both held at 260 °C, and the injector split ratio is 40:1 with a He flow rate through the column of 1.0 mL/min. Mass spectra are recorded from 60 to 300 Th at 1.5 seconds per decade (magnetic field is scanned) with a 2.5 minute solvent delay.
  • 300 < MW < 450 Da: 1 µL of the liquid is placed into an aluminum crucible and allowed to dry. The crucible is placed on the direct insertion probe (DIP) and introduced into the source of the mass spectrometer. The crucible is heated to a temperature sufficient to volatilize the compound in question (up to 250 °C). Mass spectra are recorded from 90 to 600 Th at 3 seconds per decade (magnetic field scan).
  • MW > 450 Da: 0.5 µL of the liquid is deposited onto the tungsten wire of the direct chemical ionization (DCI) probe. After drying, the DCI probe is introduced in the source and the wire is heated to volatilize the analyte (up to 1400 °C, if required). Mass spectra are recorded either by scanning the magnetic sector from 90 to 600 Th (or higher if necessary) @ 3 seconds per decade (BSCAN) or scanning the electric sector across a narrow range (typically < 100 Th) near the mass of the analayte compound at 10 seconds per decade (ESCAN).

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High Resolution Electrospray (small molecules)

Samples are diluted 3:750 in methanol containing 0.5% v/v acetic acid and 5 µmol/L sodium acetate. Three microliters of this solution are then introduced into the Waters LCT mass spectrometer using a Waters CapLC autosampler and pump. Mass spectra are recorded from 150 to 2300 Th at 1 second integration per scan. The LCT is calibrated daily using sodium iodide clusters [(NaI)nNa+]. A "lock spray" dual sprayer source enables internal calibration without mixing the calibrant with the analyte solution. Mass spectra from the total ion chromatogram (TIC) trace are combined, smoothed (two 4-point Savitzky-Golay smooths), centroided (a TDC dead-time correction is applied at this step), and internally calibrated using the 1222.1438 Th NaI cluster ion introduced through the reference inlet of the lock-spray source. Formula matching is facilitated by the Elemental Composition module of the MassLynx 4.1 software package.

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MALDI-TOF

There are a handful of procedures for MALDI-TOF depending on the type of analyte. All mass spectra are recorded on a Bruker Autoflex III mass spectrometer.

  • Small molecules and peptides (CCA mix procedure): One microliter of the sample is combined with 5 µL of a 5 g/L solution of α-cyano-4-hydroxycinnamic acid (CCA, purchased from Waters Corp) in 10:9:1 water:acetonitrile:acetone containing 0.1% trifluoroacetic acid. 1 µL of this solution is deposited onto the target and allowed to dry. The target is introduced into the mass spectrometer and irradiated with a 355 nm laser at an intensity sufficient to just ionize the sample. Mass spectra are recorded from 700 to 4,000 Th (or 379-2,100 Th) and calibrated externally using 4700 mix.
  • Small molecules (CCA overlayer procedure): A 1 uL aliquot of 10 g/L CCA mixed as described above is dried on the target. 0.5 uL of sample solution (usually in dichloromethane or chloroform) is deposited on top of the CCA spot and allowed to dry. The sample is then analyzed as described above. For internal calibration, the CCA spot contains 4700 mix. Alternatively, the sample can be applied to the target and allowed to dry BEFORE the matrix solution is applied on top of the dried sample film.
  • Large peptides/small proteins: 1 µL of the sample solution is mixed with 5 µL of 10 g/L α-cyano-4-hydroxycinnamic acid in 10:9:1 acetonitrile:water:acetone with 0.1% trifluoroacetic acid. 1 µL of the resulting mixture is deposited onto the target and allowed to dry. If internal calibration is requested, a second spot is made by adding 1 µL of 1:5 Protein Mix F:CCA matrix solution to the matrix:sample mixture. 1 µL of this new solution is allowed to dry on the target. The spot(s) is irradiated with a 355 nm laser at an intensity sufficient to just ionize the analyte. The mass spectrum is recorded (2000 shots) and the peaks from protein mix F are used to internally calibrate the data if present.
  • Polyaromatic hydrocarbons:  a few crystals of dry analyte powder are mixed with a 100-fold mass excess of tetracyanoquinodimethane (TCNQ) matrix in a 0.6 mL centrifuge tube.  Three 1.0 mm diameter stainless steel spheres are added and the tube is vortexed for 10 minutes to facilitate grinding and mixing of the powders.  A few crystals of the resulting mixture are placed onto the stainless steel target and analyzed by MALDI.  

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Low Resolution ESI (small molecules)

Samples are diluted 3:750 in methanol containing 0.5% v/v acetic acid and 5 µmol/L sodium acetate. Two microliters of this solution are then analyzed using the Agilent 1200/6130 HPLC-MS instrument.  Usually, either a mixed mode loop injection (both ESI and APCI spectra are recorded) or a 10 minute HPLC-mixed mode MS method is employed.  Mass spectra are recorded from 150 to 1000 Th at 2 second integration per scan. Data are then hand-integrated and printed out as pdf files. Positive ion mode is used unless otherwise specified by the client.

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Low Resolution ESI (biological molecules)

5 µL aliquots of the sample are mixed with 25 µL of water, placed into autosampler vials and introduced into the source of a Waters LCT mass spectrometer after separation on a 0.5 mm i.d., 150 mm long Agilent Zorbax 300SB-C18 column with a Waters CapLC programmed with one of three gradients listed below. Mass spectra are recorded from 600 to 2,300 Th with 1 second of integration time per scan. The mass spectrometer is calibrated each morning using sodium iodide clusters. The distributions of multiply charged peaks are deconvoluted to Z=0 mass spectra using ProTrawler from BioAnalyte corporation. Common gradients are:

  • Ballistic: 15 uL/min flow rate, 15% B for 0.5 min to 85% B at 2.0 min, hold at 85% B for 2.5 min, then back to 15% B for 3 min (7 min total run time)
  • 20 min: 15 uL/min flow rate, 15% B for 0.5 min, to 85% B at 12 min, hold at 85% B for 4 min, back to 5% B for 4 min (20 min total run time)
  • 45 min: 15 uL/min flow rate, 15% B for 0.5 min, to 85% B at 30 min, hold at 85% B for 6 min, back to 15% B for 9 min (45 min total run time)
  • Solvent A is 0.1% formic acid in 95% water, 5% acetonitrile
  • Solvent B is 0.1% formic acid in 95% acetonitrile, 5% water

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Low Resolution GC-MS

Solution is diluted with a few drops of dichloromethane or chloroform and then analyzed on an Agilent 6890/5973 GC-MS. 2 µL of the solution are injected onto a 0.25 mm i.d., 30 m long Agilent DB-5MS column; the following temperature gradient is used for the separation: 40 °C for 1.5 minutes, 10 °C/min to 300 °C, hold for 10 min @ 300 °C. The injector port and transfer line are both held at 280 °C, and the injector split ratio is 40:1 with a He flow rate of 1.2 mL/min through the column. Mass spectra are recorded from 50 to 450 Th at 3.7 scans per second with a 3.0 minute solvent delay.