Rapid MALDI mass spectrometry imaging for surgical pathology.

Matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) is an emerging analytical technique, which generates spatially resolved proteomic and metabolomic images from tissue specimens. Conventional MALDI MSI processing and data acquisition can take over 30 min, limiting its clinical utility for intraoperative diagnostics. We present a rapid MALDI MSI method, completed under 5 min, including sample preparation and analysis, providing a workflow compatible with the clinical frozen section procedure.

Implementation of a Gaussian beam laser and aspheric optics for high spatial resolution MALDI imaging MS.

We have investigated the use of a Gaussian beam laser for MALDI Imaging Mass Spectrometry to provide a precisely defined laser spot of 5 µm diameter on target using a commercial MALDI TOF instrument originally designed to produce a 20 µm diameter laser beam spot at its smallest setting. A Gaussian beam laser was installed in the instrument in combination with an aspheric focusing lens. This ion source produced sharp ion images at 5 µm spatial resolution with signals of high intensity as shown for images from thin tissue sections of mouse brain.

Pre-steady-state kinetics of Ba-Ca exchange reveals a second electrogenic step involved in Ca2+ translocation by the Na-Ca exchanger.

Kinetic properties of the Na-Ca exchanger (guinea pig NCX1) expressed in Xenopus oocytes were investigated with excised membrane patches in the inside-out configuration and photolytic Ca(2+) concentration jumps with either 5 mM extracellular Sr(2+) or Ba(2+). After a Ca(2+) concentration jump on the cytoplasmic side, the exchanger performed Sr-Ca or Ba-Ca exchange. In the Sr-Ca mode, currents are transient and decay in a monoexponential manner similar to that of currents in the Ca-Ca exchange mode described before. Currents recorded in the Ba-Ca mode are also transient, but the decay is biphasic. In the Sr-Ca mode the amount of charge translocated increases at negative potentials in agreement with experiments performed in the Ca-Ca mode. In the Ba-Ca mode the total amount of charge translocated after a Ca(2+) concentration jump is approximately 4 to 5 times that in Ca-Ca or Sr-Ca mode. In the Ba-Ca mode the voltage dependence of charge translocation depends on the Ca(2+) concentration on the cytosolic side before the Ca(2+) concentration jump. At low initial Ca(2+) levels (approximately 0.5 microM), charge translocation is voltage independent. At a higher initial concentration (1 microM Ca(2+)), the amount of charge translocated increases at positive potentials. Biphasic relaxation of the current was also observed in the Ca-Ca mode if the external Ca(2+) concentration was reduced to < or =0.5 mM. The results reported here and in previous publications can be described by using a 6-state model with two voltage-dependent conformational transitions.

Time resolved kinetics of the guinea pig Na-Ca exchanger (NCX1) expressed in Xenopus oocytes: voltage and Ca(2+) dependence of pre-steady-state current investigated by photolytic Ca (2+)concentration jumps.

Kinetic properties of the Na-Ca exchanger (guinea pig NCX1) expressed in Xenopus oocytes were investigated by patch clamp techniques and photolytic Ca(2+) concentration jumps. Current measured in oocyte membranes expressing NCX1 is almost indistinguishable from current measured in patches derived from cardiac myocytes. In the Ca-Ca exchange mode, a transient inward current is observed, whereas in the Na-Ca exchange mode, current either rises to a plateau, or at higher Ca(2+) concentration jumps, an initial transient is followed by a plateau. No comparable current was observed in membrane patches not expressing NCX1, indicating that photolytic Ca(2+) concentrations jumps activate Na-Ca exchange current. Electrical currents generated by NCX1 expressed in Xenopus oocytes are about four times larger than those obtained from cardiac myocyte membranes enabling current recording with smaller concentration jumps and/or higher time resolution. The apparent affinity for Ca(2+) of nonstationary exchange currents (0.1 mM) is much lower than that of stationary currents (6 muM). Measurement of the Ca(2+) dependence of the rising phase provides direct evidence that the association rate constant for Ca(2+) is about 5 x 10(8) M(-1) s(-1) and voltage independent. In both transport modes, the transient current decays with a voltage independent but Ca(2+)-dependent rate constant, which is about 9,000 s(-1) at saturating Ca(2+) concentrations. The voltage independence of this relaxation is maintained for Ca(2+) concentrations far below saturation. In the Ca-Ca exchange mode, the amount of charge translocated after a concentration jump is independent of the magnitude of the jump but voltage dependent, increasing at negative voltages. The slope of the charge-voltage relation is independent of the Ca(2+) concentration. Major conclusions are: (1) Photolytic Ca(2+) concentration jumps generate current related to NCX1. (2) The dissociation constant for Ca(2+) at the cytoplasmic transport binding site is about 0.1 mM. (3) The association rate constant of Ca(2+) at the cytoplasmic transport sites is high (5 x 10(-8) M(-1)s(-1)) and voltage independent. (4) The minimal five-state model (voltage independent binding reactions, one voltage independent conformational transition and one very fast voltage dependent conformational transition) used before to describe Ca(2+) translocation at saturating Ca(2+) concentrations is valid for Ca(2+) concentrations far below saturation.

Optimizing UV laser focus profiles for improved MALDI performance.

Matrix assisted laser desorption/ionization (MALDI) applications, such as proteomics, genomics, clinical profiling and MALDI imaging, have created a growing demand for faster instrumentation. Since the commonly used nitrogen lasers have throughput and life span limitations, diode-pumped solid-state lasers are an alternative. Unfortunately this type of laser shows clear performance limitations in MALDI in terms of sensitivity, resolution and ease of use, for applications such as thin-layer sample preparations, acceptance of various matrices (e.g. DHB for glycopeptides) and MALDI imaging. While it is obvious that the MALDI process has some dependence on the characteristics of the laser used, it is unclear which features are the most critical in determining laser performance for MALDI. In this paper we show, for the first time, that a spatially structured laser beam profile in lieu of a Gaussian profile is of striking importance. This result enabled us to design diode-pumped Nd : YAG lasers that on various critical applications perform as well for MALDI as the nitrogen lasers and in some respects even better. The modulation of the beam profile appears to be a new parameter for optimizing the MALDI process. In addition, the results trigger new questions directing us to a better understanding of the MALDI process.

Activation and inactivation kinetics of a Ca2+-activated Cl- current: photolytic Ca2+ concentration and voltage jump experiments.

The activation kinetics of the endogenous Ca(2+)-activated Cl(-) current (I (Cl,Ca)) from Xenopus oocytes was investigated in excised "giant" membrane patches with voltage and Ca(2+) concentration jumps performed by the photolytic cleavage of the chelator DM-nitrophen. Currents generated by photolytic Ca(2+) concentration jumps begin with a lag phase followed by an exponential rising phase. Both phases show little voltage dependence but are Ca(2+)-dependent. The lag phase decreases from about 10 ms after a small Ca(2+) concentration jump (0.1 microM) to less than 1 ms after a saturating concentration jump (55 microM). The rate constant of the rising phase is half-maximal at about 5 microM. At saturating Ca(2+) concentrations, the rate constant is 400 to 500 s(-1). The Ca(2+) dependence of the stationary current can be described by the Hill equation with n=2.3 and K (0.5)=0.5 microM. The amplitude of the stationary current decreases after the excision of the membrane patch with t (1/2) approximately 5 min (run-down). The activation kinetics of the current elicited by a Ca(2+) concentration jump is not affected by the run-down phenomenon. At low Ca(2+) concentration (0.3 microM), voltage jumps induce a slowly activating current with voltage-independent time-course. Activation is preceded by an initial transient of about 1-ms duration. At saturating Ca(2+) levels (1 mM), the initial transient decays to a stationary current. The transient can be explained by a voltage-dependent inactivation process. The experimental data reported here can be described by a linear five-state reaction model with two sequential voltage-dependent Ca(2+)-binding steps, followed by a voltage-independent rate-limiting transition to the open and a voltage-dependent transition to a closed, inactivated state.