• Application Note

Negative ion mode Calibration Using MassPREP Neg Ion Mode Calibrant with MALDI -TOF MS

Negative ion mode Calibration Using MassPREP Neg Ion Mode Calibrant with MALDI -TOF MS

  • Joomi Ahn
  • Ying Qing Yu
  • Martin Gilar
  • Waters Corporation


This Application note demonstrates how MassPREP Neg Ion Mode Calibrant should be prepared for MALDI MS analysis.


MALDI-TOF MS has been widely used as a very practical tool for mass analysis of biopolymers, such as peptides and oligosaccharides. Most analysis is performed in positive ion reflectron mode. Analytes such as acidic peptides, phosphopeptides and sialylated glycans have better ionization efficiency in negative ion mode, therefore, it is ideal to analyze them in the negative ion mode to obtain higher ion counts. Waters has developed a high purity calibration standard under the trade name of MassPREP Neg Ion Mode Calibrant for accurate negative ion mass calibrations. This standard provides polymeric peaks across the mass range of 700 to 4,000 amu. Sample preparation is made simple using the standard. This document demonstrates how MassPREP Neg Ion Mode Calibrant should be prepared for MALDI MS analysis. A general guideline on how to perform negative ion data acquisitions using Waters MALDI micro MX is also included.



Waters MassPREP Neg Ion Mode Calibrant (2AA-Dextran) 100μg lyophilized solids/vial

• Waters MassPREP MALDI Matrix DHB,10 mg/vial

• Ammonium tris-citrate (Sigma-Aldrich)

• Acetonitrile (High purity grade, J.T Baker)

• High purity Milli-Q water

• Waters MALDI Plate

• Glu-fibrino peptide (Glu-Fib, Sigma-Aldrich)

Calibrant Preparation

  1. Make a 1:1 mixture of acetonitrile and H2O to reconstitute the lyophilized calibrant.
  2. Add 50 μL of the solution to the vial containing the lyophilized calibrant.
  3. Vortex the vial for several seconds to ensure that the solid is dissolved.
  4. Mix equal volumes of sample solution with 2,5-dihydroxybenzoic acid (DHB) matrix solution (40 mg/mL of DHB in solution with high organic content with 8 mM ammonium tris-citrate) in a separate sample container and vortex.
  5. Apply 1 μL of the mixture onto a clean MALDI target.
  6. Dry before submitting target for MALDI analysis.
Figure 1. Calibrant Preparation.

Note: Optimization for specific applications is recommended. Other relative ratios and solvents may be used for reconstitution.

Waters MALDI micro MX

  • Acquire data in negative mode in MassLynx 4.0.
  • Run calibrant using settings below in reflectron mode.
  • Pulse Voltage =1950V

    Detector Voltage =2400V

    Flight Tube = 15,000V

    Negative Anode = 5000V

    Mass Range = 700-5000 amu

    Laser Firing Rate = 10Hz, 10 shots/spectrum

  • Adjust laser power at 220 to 240. Aim the laser at different positions for “sweet” spots.
  • After acquiring the spectrum, use the peak list (Table 1) for mass calibration.
Table 1. Peak List for MassPREP Neg Ion Mode Calibrant

Glu-fibrino peptide for mass confirmation

  • Mix 20 fmol of glu-fibrino peptide with equal volume of 10 mg/mL DHB.
  • Spot 1 μL glu-fibrino peptide mix on four neighboring wells of lock mass where the calibrant was calibrated.
  • Acquire data in negative ion mode under the same condition after calibration.

Results and Discussion

A MALDI mass spectrum of the calibrant is shown in the following page (Figure 2). The mass accuracy of the instrument after calibration is shown in Figure 3 using glu-fibrino peptide as a test sample.

Figure 2. MALDI-TOF MS spectrum of Waters MassPREP Neg Ion Mode Calibrant in negative ion mode between 700 -4000 amu
Figure 3. MALDI-TOF MS spectrum of Glu-Fib peptide after applying the calibration; (M-H)theoretical =1568.677 m/z. Calculated RMS error (n=4) was 24 ppm.

Ordering Information

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720002340, September 2007

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