Delayed extraction mass resolution

Delayed extraction and a reflectron each provide about 10-fold increases in the resolution of MALDl-TOF instruments. Delayed extraction improves resolution from -200 to -2,000, and incorporation of a reflectron increases resolution from -2,000 to -20,000. As resolution increases, so does the ability to accurately calibrate the mass scale. In linear mode (i.e., without a reflectron), only nominal mass measurements can be obtained. Accurate mass determinations become possible once a reflectron is incorporated. 

State-of-the-art ToF-MS employs reflection lenses and delayed extraction [176] to improve resolution by minimising small differences in ion energies, and in these cases up to 12000 mass resolution (FWHM, m/z 600) is available. This is sufficient for most modern applications. Solid probe ToF-MS (or direct inlet high-resolution mass spectrometry, DI-HRMS) is a breakthrough. DIP-ToFMS is a thermal separation technique. Advantages of DIP-ToFMS are ... 

Applications MALDI-ToFMS is at its best as a rapid screening technique for quick identification of known additives. However, this screening is rendered slightly more complicated by the fact that MALDI-ToFMS spectra of pure additives and of additives in the presence of excess macromolecules are not always identical (matrix effect) [55]. For unknown additives, the relation MALDI-ToFMS spectrum-chemical structure is not easily established, and the use of FD or MALDI-MS/MS is then needed. As MALDI-MS shows a sensitivity difference for the various additives, it cannot easily quantify them unless the analytes are very similar. For differentiation of additives with the same mass number (e.g. Tinuvin 315 and Cyasorb UV3638 with m/z = 368) high resolution is required, as provided by delayed extraction MALDI-ToFMS. 

If delayed extraction increases the mass resolution without degradation of sensitivity compared with continuous extraction, it also has limitations. Indeed, delayed extraction complicates the mass calibration procedure. It can only be optimized for part of the mass range at a time and is less effective at high mass. Delayed extraction partially decouples ion production from the flight time analysis, thus improving the pulsed beam definition. However, calibration, resolution and mass accuracy are still affected by conditions in the source. For instance, in the usual axial MALDI-TOF experiments, optimum focusing conditions depend on laser pulse width and fluence, the type of sample matrix, the sample preparation method, and even the location of the laser spot on the sample. 

In the example of ions A, B, and C above, by delaying the extraction voltage relative to ion formation, the ions are allowed to expand, as a function of their inherent velocities, within the source under field-free conditions. Ion A therefore moves away from the extraction lens, ion C moves toward the extraction lens, and ion B remains stationary When the extraction voltage is applied, ions A and C will have moved to regions of higher and lower extraction potential respectively, relative to their initial positions within the source (see Fig. 20b). Because of this, the turnaround time for ion A will decrease, and the additional voltage push for ion C will be minimized. This ultimately improves mass resolution because the ion source residence time differentials between A, B, and C are minimized. 

LL Haney, DE Riederer. Delayed extraction for improved resolution of ion/surface collision products by time-of-flight mass spectrometry. Anal Chim Acta 397 225-233,1999. 

The MALDI-TOF mass spectra of the Sephadex-water/ethanol (1 1) eluate (fraction 2) of both the spray ed cranberry juice and cranberry fruit showed a series of anthocyanins cyanidin-pentoside m/z 419.3), peonidin-pentoside m/z 433.3), cyanidin-hexoside m/z 449.3) and peonidin-hexoside m/z 463.3) (Figure 2A). MALDI-TOF MS equipped with delayed extraction provide unit mass resolution, allowing for the visualization of isotopic distribution. The reported observed masses (m/z) correspond to the monoisotope of the predicted compound. For example the predicted and observed monoisotope of cyanidin-pentoside is m/z 419.3) representing the contribution of C, H and 0 to the compound. The mass at m/z 420.3) represents one C, or one H, or one The mass at m/z 421.3) represents two or one C and one H, or one 0, or two H. Mass calculating programs such as IsoPro 3.0 [Shareware at http //members.aol.com/msmsoft] can be used to predict the isotopic distribution of compounds and allow for comparison between predicted and observed isotopic distributions. 

Fig. 3.5. Schematic representation of a time-of-flight mass analyser with reflectron and external ion storage for time-delay extraction. Ions that enter the field-free drift region migrate to the detector at a rate that is dependent on their m/z ratio. The reflectron lenses compensate for variations in kinetic energies of the injected ions these variations would otherwise produce broadened peaks and loss of spectral resolution.

Time-of-flight reflectron mass spectrometry with some form of time-delay extraction has become popular for large-molecule characterizations. Resolutions of up to 15,000 can be routinely achieved over very broad mass ranges, extending up to about 300,000 Da. As noted previously, time-of-flight analysers are particularly compatible with laser-based ionization techniques that produce very short bursts of ions. They are also very fast, with mass spectra often obtainable in about 25 p,s. Finally, time-of-flight analysers have been paired with a quadrupole to produce a hybrid two-dimensional mass spectrometry system that has found widespread use in protein analyses. This device will be discussed in more detail in the section on two-dimensional mass spectrometry. 

A significant improvement in mass resolution, respect to the above described "continuous extraction" procedure, was obtained by the introduction of "time lag focusing" or "delayed extraction" (DE). This principle can be used in both linear and reflection mode yielding an improvement in resolution by a factor of above ten in both cases. 

The high mass resolution that can be obtained by delayed extraction MAEDI-TOF has permitted the characterization of two poly(ethylene glycol) derivatives of pharmaceutical significance, PEG-bis(ephedrine) and PEG-bis(acefaminophenone). MALDI analysis confirmed that these polymers have ephedrine or acetaminophenone linked at both ends. End-group characterization, by MALDI-TOF, of derivatized and underivatized samples of octylphenol-teminated PEG revealed that the addition of carboxylic ends substantially enhances the detectability of these ethoxylated polymers. ... 

Vitalini, D., Mineo, R, and Scamjxjrrino, E., Effect of Combined Changes in Delayed Extraction and Potential Gradient on the Mass Resolution and Ion Discrimination in the Analysis of Polydisperse Poymers and Polymer Blends by Delayed Extraction Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry, Rapid Comm. Mass. Spectrom., 13, 2511,1999. 

Further performance improvements in analysing nucleic acids could be achieved by the introduction of 3-hydroxypicolinic add as matrix [8] and the introduction of delayed extraction in a linear time-of-flight mass spectrometer [9]. If, for MALDI Fourier transform mass spectrometry, the molecular weight range in analysing nucleic add fragments could be extended further this type of MALDI MS would become of significant value due to the extraordinary resolution possible [10, 11]. In order to reach the sensitivity level necessary for MALDI-TOF MS analysis an amplification step has to be incorporated into the sample preparation process for... 

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 major recent improvement in terms of mass accuracy and peak resolution is certainly the combination of the reflecting analyser with the delayed extraction mode [76]. They were able to achieve mass measurement accuracy of 2 ppm on a 1000 Da peak with a resolution as high as 10.000 FWHM. Such improvement in accuracy has obvious implications in the reliability of protein identification using PMF. Higher resolution can be obtain when MALDI-MS is coupled with a Fourrier Transform Ion Cyclotron Resonance (FTlCR) but the major drawback of such system is its expense. 

Reflectrons (ion mirrors) are used to increase the resolution, as well as the ability to obtain accurate mass measurements, by counteracting the effect of the energy spread not compensated for by collisional cooling at the delayed extraction grid (in MALDI sources). A reflectron is a cylinder composed of concentric rings and grids... 

A TOP mass analyzer separates ions on the basis of their velocity differences. A short pulse of ions of a defined kinetic energy is allowed to drift in a long field-free flight tnbe in which the lighter ions travel faster and reach the detector earlier than do the heavier ones. Some innovative developments are incorporated into the linear TOP design to improve resolution (e.g., the addition of a reflectron and the use of delayed extraction of ions) and make it suitable for a continuous mode of ion beams. 

Other types of mass spectrometer have been used to record MALDI spectra magnetic sector instruments equipped with an array detector initially offered the advantage of higher resolution than was available with the early linear TOF instruments but these spectrometers have now largely been replaced with reflectron-TOF instruments using delayed extraction ion sources. Mass spectrometers operated with higher than normal ion source pressures to... 

The introduction of delayed ion extraction in MALDI-TOF instruments in 1995 resulted in a tremendous improvement in analytical performance, mass resolution and accuracy. Using this technique, peptide mass mapping gained high specificity for protein identification and an ability to identify individual components among simple protein mixtures [60, 61]. Improved sample preparation methods also provided an enhanced sensitivity to enable analysis of femtomolar levels of protein [5, 19], Today, the automation of MALDI-MS data acquisition enables the analysis of hundreds of samples each day in large-scale protein identification experiments [62]. 

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