This instrument combines the capabilities of ion/ion reactions with ion mobility (IM) and time-of-flight (TOF) measurements for conformation studies and top-down analysis of large biomolecules. Ubiquitin ions from either of two electrospray ionization (ESI) sources are stored in a three dimensional (3D) ion trap (IT) and reacted with negative ions from atmospheric sampling glow discharge ionization (ASGDI). The proton transfer reaction products are then separated by IM and analyzed via a TOF mass analyzer. In this way, ubiquitin ϩ7 ions are converted to lower charge states down to ϩ1; the ions in lower charge states tend to be in compact conformations with cross sections down to ϳ880 Å 2 . The duration and magnitude of the ion ejection pulse on the IT exit and the entrance voltage on the IM drift tube can affect the measured distribution of conformers for ubiquitin ϩ7 and ϩ6. Alternatively, protein ions are fragmented by collision-induced dissociation (CID) in the IT, followed by ion/ion reactions to reduce the charge states of the CID product ions, thus simplifying assignment of charge states and fragments using the mobility-resolved tandem mass spectrum. Instrument characteristics and the use of a new ion trap controller and software modifications to control the entire instrument are described. Ion mobility (IM) [4,5] has become a very useful technique for analysis of biological ions in the gas phase [6]. IM provides information about ion size and structure [7], as it rapidly separates ions based on collision cross-section, rather than just m/z ratio. The use of IM to disperse a mixture of ions in time before analysis via a time-of-flight (TOF) MS, i.e., nested drift (flight) time measurements, is an important recent advance. These experiments were pioneered by Clemmer and coworkers in the mid-1990s [8], and have now been used by several other groups [9 -13]. The recent Synapt ESI-IMS-MS by Waters Corp. provides a commercially available instrument for gas-phase ion conformation studies by IM using a novel traveling wave approach [9].In a series of instrumental designs, the Clemmer group has made various modifications to the initial ESI-IM-TOF, including the insertion of a collision cell between the IM drift tube and the TOF for mobility labeling experiments [14,15], and the addition of an IT before the mobility drift tube to improve the duty cycle from the continuous ESI source [16,17]. One publication demonstrated MS/MS capabilities with an ion trap before IM-TOF [18], but the entire instrument was not under computer control. Therefore, only relatively simple experiments were possible.IM has been used to analyze the products of ionmolecule reactions [19], including proton transfer [20,21], H/D exchange [22], and solvation [23][24][25][26]. In these studies, the desired reactions take place either in the atmospheric pressure ion source interface region or in the drift tube itself. Thus, only short reaction times and certain reagent ions can be used. In addition, performing reactions in the IM cell can make spe...

Positive ions from cytochrome c are studied in a 3-D ion trap/ion mobility (IM)/quadrupoletime-of-flight (TOF) instrument with three independent ion sources. The IM separation allows measurement of the cross section of the ions. Ion/ion reactions in the 3-D ion trap that remove protons cause the cytochrome c ions to refold gently without other degradation of protein structure, i.e., fragmentation or loss of heme group or metal ion. The conformation(s) of the product ions generated by ion/ion reactions in a given charge state are similar regardless of whether the cytochrome c ions are originally in ϩ8 or ϩ9 charge states. In the lower charge states (ϩ1 to ϩ5) cytochrome c ions made by the ion/ion reaction yield a single IM peak with cross section of ϳ1110 to 1180 Å 2 , even if the original ϩ8 ion started with multiple conformations. The conformation expands slightly when the charge state is reduced from ϩ5 to ϩ1. For product ions in the ϩ6 to ϩ8 charge states, ions created from higher charge states (ϩ9 to ϩ16) by ion/ion reaction produce more compact conformation(s) in somewhat higher abundances compared with those produced directly by the electrospray ionization ( [5][6][7][8][9], and native electron capture dissociation (NECD) [10,11]. Of these methods, IM provides a direct way to examine the gas-phase conformation of the ions by probing the average cross-section of the protein ions via collisions with buffer gas [3,4]. Early IM research on protein folding and unfolding was done with an IMquadrupole instrument [1]. To study ions in lower charge states than those made directly from the electrospray ionization (ESI) source, a basic collision gas (e.g., acetophenone or 7-methyl-1,3,5-triazabicyclo 4,4,0] dec-5-ene, MTBD) [12] was introduced into the source region. The neutral gas extracted protons from the protein ion and created lower charge state ions through proton transfer reactions in the source. In these studies, the reactions took place only in the atmospheric pressure ion source interface region. Thus, control and variation of the reaction time and extent of reaction were difficult, and only certain reagent species were available.Gas-phase ion/ion reactions provide another dimension for gas-phase bioanalysis by MS. To date, these reactions have been mainly used to simplify complex MS/MS spectra [13,14] or generate fragments for structural assignment [15,16] by methods like electrontransfer dissociation (ETD) [17][18][19].Recent instrumentation improvements have greatly extended the type of structural information and number of possible experiments available in this area. The development of a 3-D trap-IM-time of flight (TOF) instrument allows time-dependent studies of gas-phase protein ions, including folding, unfolding and structural transitions [20 -23]. A multi-stage IMS-MS instrument [24,25] provides two important new functions. First, a protein ion with a specific structure can be selected by IMS, then activated and separated in the next drift region. Second, "state-to-state" structural

Colloidal graphite is a promising matrix for atmospheric pressure laser desorption/ionization mass spectrometry. Intact [M+H](+) and [M-H](-) ions are readily produced from a wide range of small molecule plant metabolites, particularly anthocyanins, fatty acids, lipids, glycerides, and ceramides. Compared with a more traditional organic acid matrix, colloidal graphite provides more efficient ionization for small hydrophobic molecules and has a much cleaner background spectrum, especially in negative ion mode. Some important metabolites, e.g., fatty acids and glycosylated flavonoids, can be observed from Arabidopsis thaliana leaf and flower petal tissues in situ.

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We report results for a new gas chromatography detector that is comparatively sensitive and far more selective for aromatic compounds than the traditional photoionization detector. The detection means is multiphoton ionization at atmospheric pressure. The ionization source in these experiments is a diode-pumped passively Q-switched microchip laser operating at 266 nm. Experiments were conducted with the detector interfaced to a fast gas chromatograph. For <20 s elution time, limits of detection were <1 pg for toluene, ethylbenzene, xylenes, and isopropylbenzene; the limit of detection for benzene is approximately 10 pg. Detector response was linear over 5 orders of magnitude, including these low levels. Negligible signals were observed for nonaromatic ketones, aldehydes, ethers, and cycloalkanes at levels as high as 0.1 microg (10 mg/L concentration). Detector efficiency after fast GC separation was 0.002% when using a detector cell with a radius of 1.1 cm and a purge gas flow of 500 mL/min. The advantages of this detector are further illustrated by the fast GC analysis of fuel samples.