A new technique has been developed that provides affinity separations directly on the surface of a matrix-assisted laser desorption/ionization mass spectrometer (MALDI-MS) probe. This strategy provides both in situ identification of biomolecules through biospecific antibody/antigen interactions and molecular weight information in a rapid and facile manner. Our technique, which we call probe affinity MS, directly couples the high selectivity of immobilized affinity chromatography with the sensitivity of MALDI-MS. As this technique permits the rapid identification of antigens, ligands, and other compounds from complex biological mixtures, we believe that it will be useful in a wide range of applications in virtually all fields of the life sciences.

Purpose: A phase1study to determine the maximum-tolerated dose, dose-limiting toxicity, pharmacokinetics, and biological effects of bortezomib in children with recurrent/refractory leukemia. Experimental Design: Bortezomib was administered twice weekly for 2 consecutive weeks at either 1.3 or 1.7 mg/m 2 dose followed by a 1-week rest. Bortezomib pharmacokinetics and nuclear factor nB (NF-nB) binding activity were evaluated during the first treatment cycle. Results:Twelve patients (nine with acute lymphoblastic leukemia, three with acute myelogenous leukemia), median age 11years (range, 1-18 years), were enrolled between May 2004 and November 2005, of whom seven were not fully evaluable for toxicity due to rapidly progressive disease or uncontrolled infection. Dose-limiting toxicities occurred in two patients at the 1.7 mg/m 2 dose level. One patient experienced grade 3 confusion and the other patient had grade 4 febrile neutropenia associated with grade 4 hypotension and grade 3 creatinine. Pharmacokinetic analysis at 1.3 mg/m 2 revealed a clearance of 11 mL/h/m 2 , a central volume of distribution of 6.7 L/m 2 , and a terminal half-life of 12.6 h. NF-nB activity was examined in five patients and was noted to transiently increase and then decrease 4-to 6-fold by 24 h following bortezomib in two patients. There were no objective clinical responses. Conclusions: For children with leukemia, the recommended phase 2 dose of bortezomib, administered twice weekly for 2 weeks followed by a 1-week rest, is 1.3 mg/m 2

One of the problems encountered in preparing samples for matrix-assisted laser desorption/ionization (MALDI) analysis is the presence of nonvolatile salts in the sample. This difficulty is often exacerbated by the necessity to prepare the sample in the appropriate sample-to-matrix ratio. This paper reports a probe surface derivatization method that greatly simplifies this sample preparation process. By constructing self-assembled monolayers of octadecyl mercaptan (C18) on the MALDI probe surface, we were able to generate a surface capable of reversibly binding polypeptides via hydrophobic interactions, which in turn, permits the analyte to be easily concentrated and desalted directly on the probe tip.

Probe affinity mass spectrometry (PAMS) is a technique that combines affinity separations directly with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In this approach, a binding molecule, such as an antibody, lectin or receptor, is covalently attached to the surface of a MALDI probe. This permits the analyte of interest to be selectively captured and concentrated on the probe surface prior to MALDI-MS analysis. A major limitation of our initial PAMS immobilization chemistry was that it produced only a relatively small number of binding sites on the probe, as it was based on forming a single monolayer of the binding molecule. Because of this limitation, we have investigated new immobilization chemistries for PAMS that are not confined by monolayer formation and thus allow a larger number of analyte molecules to be captured by the probe. We have developed a new PAMS chemistry that first attaches very high molecular weight (approximately 500,000) dextrans to the MALDI probe, followed by immobilization of the binding molecules to the probe-bound dextrans. Because the size of each dextran molecule is significantly larger than the binding molecule, multiple binding molecules can be linked to the same dextran chain. We have demonstrated that these surfaces possess approximately 500 times more analyte binding sites than probes prepared with our original PAMS chemistry. This chemistry is applicable to any binding molecule that contains a primary amine and is suitable, therefore, for a wide range of applications.

Characterization of polystyrene on etched silver substrates has been carried out using ion scattering (ISS) and X-ray photoelectron spectroscopy (XPS). The results have been used to describe the variation of time-offlight secondary-ion yield (TOF-SIMS) for polystyrene as a function of coverage. It was determined that the TOF-SIMS secondary-ion yield increases with surface coverage up to ca. 0.50 and then drastically decreases for coverages greater than 0.55. XPS results also indicate that polystyrene forms a multilayer structure, a minimum of 5 layers thick on the Ag surface over the concentration range investigated (1 pg/mL to 50 mg/mL) .