MALDI accelerating voltage

Tang, X. Dreifuss, P.A. Vertes, A. New Matrixes and Accelerating Voltage Effects in MALDI of Synthetic Polymers. Rapid Commun. Mass Spectrom. 1995, 9, 1141-1147. 

MALDI-TOF was done on a Kratos Kompact Maldi III mass spectrometer fitted with a standard 337 nm nitrogen laser and operated in the linear mode at an accelerating voltage of 20 kV. The matrix used was a-cyano-4-hydroxyciimamic acid (33mM in acetonitrile/methanol, premade from BRS) at a ratio of 1 1 with purified peptide samples. MALDI sample slides were loaded with O.S-1.0 uL of matrix/sample mixture (estimated 1-10 pmol peptide). The data was reprocessed using the Kratos software provided with the instrument. Theoretical masses were determined by utilizing a spreadsheet in which individual peptide masses were added to all possible caibohycfrate forms these masses were then compared to the observed masses to identify structures consistent with the mass results obtained. 

MALDI-MS was performed using a Kratos Kompact MALDI III mass spectrometer fitted with a standard 337 nm nitrogen laser, and operated in the linear mode at an accelerating voltage of 20 kV. Two sample preparation methods were used (1) for collected peptides, 0.3 pL aliquots of sample and matrix (a-cyano-4-hydroxy cinnamic acid, Biomolecular Separations, Inc.) were mixed on the probe slide and allowed to air dry or (2) for unfiactionated digests, a thin polycrystalline film was prepared according to (11) (with modifications for use on a probe slide (12)), matrix and sample aliquots were mixed (usually 0.3 pL each) on this surface and prior to drying, rinsed twice with 2 pL of deionized water. 

MALDI TOP (7) mass spectra were recorded on a Bruker Reflex instrument (Billerica, MA). The rp-HPLC fractions containing PMP labeled oligosaccharides were dried in a SpeedVac concentrator (Farmingdale, NY) and redissolved in water/acetonitrile (75/25, v/v). 2,5-Dihydroxybenzoic acid (DHB) was used as a matrix. Normally, 0.3 uL of a half-saturated solution of DHB in water/acetonitrile/trifluoroacetic acid (70/30/0.1, v/v) was mixed on the sample dish with 0.3 pL of the sample solution. Desorption and ionization was done with a nitrogen laser (337 nm) adjusted to minimum laser attenuation. Mass spectra (10 to 20) were accumulated in linear negative ionization mode with an acceleration voltage of 25 kV. Calibration was done externally with bovine insulin and angiotensin I. 

Mass spectra were collected on a Bruker Reflex II-MALDI-TOF mass spectrometer (Billerica, MA) equipped with delayed extraction and a N2 laser (337 nm). In the positive reflectron mode, an accelerating voltage of 25.0 kV and a reflectron voltage of 26.5 kV were used. Spectra are the sum of 50-300 shots. Spectra were calibrated with bradykinin (1060.6 MW) and glucagon (3483.8 MW) as external standards. 

Figure 6. Peptide mass profiling of a silver stained protein spot from a narrow range, pH 4-7, 2DE separation of human heart (ventricle) proteins. A MALDI-TOF mass spectrum of tryptic peptides is shown, analyzed using a Micromass Tofspec 2E spectrometer (Manchester, UK) operated in the positive ion reflectron mode at 20 kV accelerating voltage with time-lag focusing enabled. The protein spot of interest was identified as human vimentin, (courtesy of J.A.Westbrook and R. Wait) (unpublished data). 2DE gels were silver stained using a modified Amersham Biosciences kit.

A schematic TOF mass spectrometer is shown in Fig. 9.20. The drift mbe in a TOF system is approximately 1 -2 m in length. Pulses of ions are produced from the sample using pulses of electrons, secondary ions, or laser pulses (e.g., MALDI). Ion pulses are produced with frequencies of 10-50 kHz. The ions are accelerated into the drift tube by a pulsed electric held, called the ion-extraction held, because it extracts (or draws out) ions into the held-free region. Accelerating voltages up to 30 kV and extraction pulse frequencies of 5-20 kHz are used. 

Others groups [71, 72, 73, 74] obtained an improvement of mass accuracy and peak resolution by the use of a delayed extraction system [75]. This system uses a pulsed ion extraction MALDI ionisation technique increasing the accelerating voltage from 0 up to 3 kV in 300 nanoseconds. This technique allowed an increase of peak resolution for cytochrome c (12 kDa) from 350 FWHM obtained in linear mode to 1024 with a continuous ion extraction. 

The silsesquioxane mixtures or the separated silsesquioxanes can be analyzed by MALDl-TOF-MS (Table 2). MALDI-TOF mass spectrometry was carried out on a Kratos Kompact MALDI 111. 0.5 pL of a solution (25 mg/mL) of 2,4,6-trihydroxyacetophenone or 20 mg/mL of 2-nitrophenyl octyl ether and 10 mg/mL silver trifluoroacetate were mixed on a stainless steel sample slide. The solvent was evaporated in a stream of air at ambient temperature. Bovine insulin was used for calibration. Conditions for the measurements polarity positive, flight path reflection, 20 kV acceleration voltage, nitrogen laser (A = 337 nm). 

---