A formidable challenge for mass spectrometry over the years as an analytical tool for biomolecular analysis has been its utility in the measurement of dynamic biological processes. Such applications often include measurements of reactants or products whose concentrations are changing rapidly with time. Most biological processes occur in aqueous solutions, some at extraordinarily low amounts in the presence of high salt concentrations, making analytical procedures difficult. Further, analyses must be sufficiently rapid to fix the chemical pattern of the system at the instant a sample is taken. The advent of electrospray (ES) and matrix-assisted laser desorption (MALD) ionization in recent years has gone a long way in solving such problems and have brought capabilities of great utility to investigators who need to measure such dynamic systems.

ES offers advantages of high sensitivity, high molecular weight, and the capability of direct analysis of aqueous solutions. We have optimized ES to provide ultra-high sensitivity at low flow rates (micro-ES) with an integrated desalting capability. Micro-ES was combined with microdialysis (Md), to provide a tool for the in situ analysis of complex and dynamic biological reactions in living animals. Md probes are implanted in animal tissue and dialysate is delivered directly to the mass spectrometer to achieve molecular analysis. This process will be illustrated with specific examples, including the pharmacokinetics of exogenously administered therapeutic drugs, the metabolism of natural compounds and intermediates in specific tissues, and the measurements of released physiologically active molecules at subfemtomole levels. MALD mass spectrometry may also be used to analyze ongoing biochemical reactions. We have used this technique in the analysis of time-course proteolysis of peptides and proteins to reconstruct a 'sequence-ordered' proteolytic map of the products. In addition, we have measured the subunit structure of multienzyme complexes and their assembly pathways in quantitative measurements up to about 250 k Da. In the latter application, cross-linking reagents are used to trap associated or partially associated complexes under various experimental reaction conditions. In another application, desalting procedures have been employed to examine tissue extracts and dialysates at very high sensitivity.