We present two new desorption strategies for the mass spectrometric analysis of macromolecules. These desorption strategies are based on the molecular design and construction of two general classes of Sample 'probe' surfaces. The first class of surfaces is designed to enhance the desorption of intact macromolecules presented alone (neat) to the surface; we call this surface-enhanced neat desorption (SEND). The availability of probe surfaces derivatized with, or composed of, multiple types and defined numbers of energy-absorbing molecules will facilitate investigations of energy transfer and desorption/ionization mechanisms. The second class of probe surfaces is designed to enhance the desorption of specific macromolecules captured directly from unfractionated biological fluids and extracts; we call this surface-enhanced affinity capture (SEAC). Use of these new probe surfaces as chemical& defined solid-phase reaction centers will facilitate protein discovery through molecular recognition in situ, and also macromolecular structure analysis through the sequential chemical and/or enzymatic modification of the adsorbed analyte in situ. Specific examples of laser-assisted SEND and SEAC timeof-flight mass spectrometry are presented to illustrate the potential for increased selectivity, analyte detection sensitivity, and mass measurement accuracy.The introduction of matrix molecules to assist the laser-induced desorption/ionization (MALDI) of intact macromolecules has enabled relatively small quantities of large nonvolatile biopolymers to be evaluated by time-of-flight (TOF) mass spectrometry.' Many new types of investigations have been enabled by this technology.* The ability to measure precise differences in molecular mass enables problems associated with unknown protein structures to be solved more r e a d i l~.~.~ Thus, the future of biological mass spectrometry will be defined by simpler and more sensitive means to monitor specific changes in protein and peptide mass.Our laboratory has developed MALDI-TOF to explore various specific ligand-ligate interactions and to analyze protein structure;%" both approaches have been based largely on biochemical reactions performed directly on the probe surface (i.e., in situ) to exploit the availability of analyte not consumed by previous laserinduced desorption events. However, the required presence of a large molar excess of acidic matrix and the random deposition of embedded macromolecular analytes in and around an undefined array of matrix crystals has limited the use of the probe surface as an effective stationary phase from which to conduct, by differential mass spectrometry, a sequential in situ approach to the investigation of protein structure and function.An effective mass spectrometric strategy for protein structure analysis may be realized if each of several diagnostic alterations in mass can be brought about as a result of controlled chemical and/or enzymatic modification events performed directly and sequentially in situ, i.e., directly on the probe surface. In fa...

Insulin is released from the islets of Langerhans in discrete pulses that are linked to synchronized oscillations of intracellular free calcium ([Ca(2+)]i). Associated with each synchronized oscillation is a propagating calcium wave mediated by Connexin36 (Cx36) gap junctions. A computational islet model predicted that waves emerge due to heterogeneity in β-cell function throughout the islet. To test this, we applied defined patterns of glucose stimulation across the islet using a microfluidic device and measured how these perturbations affect calcium wave propagation. We further investigated how gap junction coupling regulates spatiotemporal [Ca(2+)]i dynamics in the face of heterogeneous glucose stimulation. Calcium waves were found to originate in regions of the islet having elevated excitability, and this heterogeneity is an intrinsic property of islet β-cells. The extent of [Ca(2+)]i elevation across the islet in the presence of heterogeneity is gap-junction dependent, which reveals a glucose dependence of gap junction coupling. To better describe these observations, we had to modify the computational islet model to consider the electrochemical gradient between neighboring β-cells. These results reveal how the spatiotemporal [Ca(2+)]i dynamics of the islet depend on β-cell heterogeneity and cell-cell coupling, and are important for understanding the regulation of coordinated insulin release across the islet.

A method of protein quantitative analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is described. To decouple the quantitative approach from the extreme dependence of absolute ion signal on the experimental parameters of the matrix-assisted technique, the method relies

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ABSTRACT. The origin of intact (78-kD) lactoferrin found in the urine of human milk-fed preterm infants was investigated using human milk containing proteins enriched with [13Cjle~cine and ['5N2]lysine or [2H4]lysine. Mothers of infants selected for the study were infused i.v. with [I3q leucine and ["N2]lysine or [2H4]lysine to label milk proteins. The labeled milk was collected from each mother, pooled, fortified with a lyophilized human milk fraction, and fed to her preterm infant by continuous orogastric infusion for a period of 48 h. Urine was collected from each infant for 96 h. Intact lactoferrin (78 kD) and DNAbinding lactoferrin fragments (51 and 39 kD) were purified from the urine by affinity chromatography on columns of immobilized single-stranded DNA-agarose. The concentration and isotopic enrichment of the intact lactoferrin and DNA-binding fragments were determined separately after their isolation by high-performance reverse-phase (phenyl) chromatography. Mass spectral analyses indicated that the isotopic enrichment of the purified urinary lactoferrin was 87 to 100% of that in the labeled human milk lactoferrin. Similar results were obtained for the isolated DNA-binding lactoferrin fragments. The ratios of isotopically labeled leucine to lysine in the purified milk lactoferrins and urinary lactoferrins were similar for each motherfinfant pair. Isotopically labeled lysine, added to the milk as free amino acid, was not incorporated into the purified urinary lactoferrin. These results demonstrate that undegraded (78-kD) lactoferrin of maternal origin is absorbed by the gut and excreted intact in the urine of preterm infants; nearly all of the urinary lactoferrin was of maternal origin. Lactofemn is a major constituent of human colostral milk and whey (I). This 78-kD secretory glycoprotein binds several different transition metal ions (2, 3), but is best characterized for its iron-binding properties and its association with host defense at mucosal surfaces (4). Perhaps more interesting are its postulated growth-promoting (5,6) and immune-modulating properties (7-1 1). The structures of human hololactofemn and apolactofemn are known (12, 13), but their full biologic significance and mechanisms of action remain the subject of continued investigations.Utilization of lactofemn by the immature gastrointestinal system of preterm and term infants is poorly characterized. Infants fed human milk excrete higher levels of fecal lactofemn than do infants fed bovine milk-based formulas (14-18). Fecal excretion, however, accounts for only a few percent (1-6%) of the lactofemn fed ( 16,17), and most fecal lactofemn is degraded to lower molecular mass forms (1 9-2 1). Investigators have also reported higher levels of lactofenin and lactoferrin fragments, free secretory component, and secretory IgA in the urine of infants fed human milk than in the urine of infants fed a bovine milk-based formula (21,22). The molecular sizes of these immune factors are larger than normally filtered by glomeruli (23). The serum levels of...