Fundamental studies of MALDI with an orthogonal TOF mass spectrometer

The interaction between the matrix and analyte molecules are studied with a high resolution MALDI imaging technique in an orthogonal-injection time of flight (TOF) mass spectrometer. The analyte incorporation and distribution patterns have been clearly demonstrated. Purified protein analytes were found to be homogeneously incorporated in large single crystals of DHB and sinapinic acid matrices, with no evidence for preferred crystal faces. Segregation of some species was observed and appeared to correlate with analyte hydrophobicity, and to a lesser extent analyte mass or mobility. Similar segregation phenomena were observed with confocal laser scanning microscopy of the same analytes labeled with fluorescent dyes in 2,5-DHB single crystals. The above investigations may shed some light on optimizing sample preparation with different matrices.;Finally the detection properties of the MCP detector were studied with a hybrid MCP and CuBe venetian blind converter detector. The measurements show that the detection efficiency of the MCP drops with the increasing of ion mass and the decreasing of the ion energy. For transferrin (79,500 Da), the relative detection efficiency of the MCP is about 40% at 10.6 keV and it decreases to about 5% at 4.6 keV. The secondary electron emission coefficient of the MCP shows a linear dependence on mass and a power law dependence on velocity (∼3.2). No clear velocity threshold is observed for secondary electron emission.;The influence of incident laser parameters on sensitivity in MALDI has been investigated using orthogonal-injection TOF instruments. A qualitative comparison was first made between the beam profiles obtained with a N 2 laser and a Nd:YAG laser using 2-m long optical fibers. The N 2 laser gives better sensitivity, consistent with a more uniform fluence distribution and therefore better coverage of the N2 laser profile. Most of the difference disappears when a 30-m long fiber is used or when the fibers are twisted during irradiation to smooth out the fluence distribution. In more systematic measurements, the total integrated ion yield from a single spot (a measure of sensitivity) was found to increase rapidly with fluence to a maximum, and then saturate or decrease slightly. Thus, the optimum sensitivity is achieved at high fluence. For a fluence near threshold, the integrated yield has a steep (cubic) dependence on the spot size, but the yield saturates at higher fluence for smaller spots. The area dependence is much weaker (close to linear) for fluence values above saturation, with the result that the highest integrated yields per unit area are obtained with the smallest spot sizes. The results have particular relevance for imaging MALDI, where sensitivity and spatial resolution are important figures of merit.