Atmospheric Pressure Free Liquid Infrared MALDI Mass Spectrometry: Toward a combined ESI/ MALDI-Liquid Chromatography Interface Erdmann Rapp,* ,† Ales ˇ Charva ´ t,* ,‡ Alexander Beinsen, § Uwe Plessmann, | Udo Reichl, † Andreas Seidel-Morgenstern, † Henning Urlaub, | and Bernd Abel §,⊥ Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany, Department of Photochemistry and Kinetics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Go ¨ ttingen, Germany, Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Go ¨ ttingen, Germany, Institute for Physical Chemistry, Georg-August University of Go ¨ ttingen, Tammannstrasse 6, 37077 Go ¨ ttingen, Germany, W.-Ostwald-Institute for Physical and Theoretical Chemistry, University Leipzig, Linne ´ -Strasse 2, D-04103 Leipzig, Germany A new atmospheric pressure (AP)-MALDI-type interface has been developed based on a free liquid (FL) micro- beam/microdroplets and a mid-infrared optical paramet- ric oscillator (mid-IR OPO). The device is integrated into a standard on-line nanoESI interface. The generation of molecular ions in the gas phase is believed to be the result of a fast (explosive) laser-induced evaporative dispersion (not desorption) of the microbeam into statistically charged nanodroplets. Only the lowest charge states appear in significant abundance in this type of experiment. Mass spectra of some common peptides have been acquired in positive ion mode, and the limit-of-detection of this first prototype (liquid microbeam setup) was evaluated to be 17 fmol per second. To improve the duty cycle and to reduce the sample consumption, a droplet-on-demand system was implemented (generating 100 pL droplets). With this setup, about 20 attomole of bradykinin were sufficient to achieve a signal-to-noise ratio better than five. This setup can be operated at flow rates down to 100 nL/ min and represents a liquid MALDI alternative to the nanoESI. Our particular interest was the application of the developed ion source for on-line coupling of liquid chromatography with mass spectrometry. The flow rates (>100 μL/min), required for stable operation of the ion source in continuous liquid microbeam mode, matches perfectly the flow rate range of microHPLC. Therefore, on- line LC/MS experiments have been realized, employing a microbore C18 reversed-phase column to separate an artificial peptide mixture and tryptic peptides of bovine serum albumin (performing a peptide mass fingerprint). In the latter case, sequence coverage of more than 90% has been achieved. The analysis of biomolecules by mass spectrometry put rather severe constraints on the ionization method, as far as its ability to produce intact biomolecular ions (without uncontrollable fragmentation) is concerned. With the advent of the electrospray ionization (ESI) 1,2 and matrix-assisted laser desorption/ionization (MALDI), 3-5 two ionization sources of an unprecedented softness appeared. Because of the increasing need for powerful analytics of complex mixtures, the use of LC/MS (together with GC/MS), for example, in live sciences, airport surveillance, or food control is well established nowadays. 6-10 Concomitantly, coupling of separation methods, such as high- performance liquid chromatography (HPLC), with the mass spectrometric detection appeared prerequisite for dealing with complex mixtures of biomolecules, such as protein digests of cell lysates. The quest for the best LC/MS interface started early in the 1970s (cf. refs 11, 12 and references cited therein). Two main strategies have been pursued and developed. 13-15 First, the off- line LC/MS analysis, 14,16,17 where separation and MS acquisition are decoupled. 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