Time-lag focusing MALDI

Partial mass spectrum of cow s milk (containing 2% milk fat) observed by MALDI/time-of-flight mass spectrometry. [From R. M. Whlttal and L Li, "Time-Lag Focusing MALDI-TOF Mass Spectrometry," Am. Lab. December 1997, p. 30.]... 

Whittal, R. M., Schriemer, D. C., and Li, L., Time-lag focusing MALDI time-of-flight mass spectrometry for polymer characterization oligomer resolution, mass accuracy, and average weight information. Anal. Chem., 69, 2734, 1997. 

Improved resolution and mass accuracy have been demonstrated using time-lag focusing MALDI-ToF-MS [81]. The upper mass limit of oligomer resolution was extended using this method. However, the overall resolution of polymer spectra was still found to be dependent on the matrix used and sample preparation methods. 

Figure 2.10. Schematic of a MALDI-TOF mass spectrometer with reflector and time-lag focusing. Reprinted from A. Westman-Brinkmalm and G. Brinkmalm (2002). In Mass Spectrometry and Hyphenated Techniques in Neuropeptide Research, J. Silberring and R. Ekman (eds.) New York John Wiley Sons, 47-105. With permission of John Wiley Sons, Inc.

The mass accuracy is highly dependent on the mode the instrument is operating in. In the reflector mode, with time-lag focusing, the best MALDI-TOF and oa-TOF instruments are capable of achieving <5 ppm with internal standards, provided that the isotopes are resolved. In many cases it is not possible to add internal calibrants, and then the error in mass accuracy is often increased to 50-100 ppm. Operation of an instalment in a linear mode will typically decrease the mass accuracy. 

Time lag focusing presumes that (a) ionization occurs within the source and (b) the ionization mechanism imparts upon the ions a distribution of transient energies and velocity vectors. An ion source such as that used in MALDI can greatly benefit from time lag focusing, and this technology is becoming standard on commercial MALDI/TOF instruments. 

Today, the modern version of time-lag focusing, or delayed extraction,14 16 is used in almost all MALDI-TOF mass spectrometers. It is in fact a special case of the Wiley and McLaren method, in which the initial spatial distribution (usually a very thin sample and matrix mixture dried on a stainless steel plate) is assumed to be zero. Therefore, after the delay time the velocity and spatial distributions are correlated, i.e. ions of highest velocity have moved the greatest distance. This space velocity correlated focusing has been described by Colby and Riley,17 and provides considerably better focusing than can be obtained from two independent and uncorrelated initial distributions in space and velocity/energy that comprise the general Wiley-McLaren case. 

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.

The initial velocity spread is converted to a spatial spread by allowing a delay between ionization and acceleration. Ions separate to create a correlation between position and velocity. Spatial focusing can be applied to correct this. This method was also developed by Wiley and McLaren and the conditions (length of delay) must be adjusted for each miz. Time-lag focusing does not work well when a significant spatial and velocity spread are present simultaneously. As will be discussed later, time-lag focusing has recently been adapted to greatly increase the resolution in MALDI TOF-MS. 

Figure 8.9 (a) MALDI mass spectrum of poly(ethylene glycol) 20000 using HABA as matrix with sodium cationization and time-lag focusing set to optimize the resolution of ions at m/z 23000. (b) Expansion of (a) in the high mass region of the spectrum, (c) Expanded MALDI mass... 

To compensate for the energy spread, the first stage of the two-stage acceleration field can be activated shortly after ion production or ejection from the sample. This event typically occurs in the nanosecond to low microsecond range. The method is known as time-lag focusing or delayed extraction technique. In TOF-MS equipped with matrix-assisted laser desorption/ionization (MALDI) ion source, delayed extraction may improve the mass resolving power by a factor of two- to fivefold [9, 10]. Mass resolving power defines the sharpness of spectral features, which is explained in Chapter 5. 

The important instrumental development of delayed (ion) extraction (DE) or time-lag focusing (TLF) [279] has had a great impact on MALDI development [280,281]. The major limitation to the resolution provided by MALDI-MS is the initial velocity distribution of the ions. Ions with the same mass/charge ratio but different energy distributions yield a broad peak with a decrease in resolution. Correction for this by the use of pulsed extraction for time-lag focusing [282] has greatly improved the quality of the mass spectra from low masses (<100 Da) up to high masses. The term time-lag... 

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