Application of a non-indexed dual sprayer pneumatically assisted electrospray source to the high throughput quantitation of target compounds in biological fluids.

The need for increased throughput in the quantitation of target compounds in biological fluids by high performance liquid chromatography/mass spectrometry continues to drive research in this area. This report describes the application of a prototype dual sprayer electrospray source for the quantitative analysis of biological samples. Quantitative performance for 180 compounds in a microsomal stability assay was found to be adequate when compared with a conventional single sprayer measurement. Issues with use of dual sprayers in a routine production environment are discussed.

Microsatellite markers reveal a spectrum of population structures in the malaria parasite Plasmodium falciparum.

Multilocus genotyping of microbial pathogens has revealed a range of population structures, with some bacteria showing extensive recombination and others showing almost complete clonality. The population structure of the protozoan parasite Plasmodium falciparum has been harder to evaluate, since most studies have used a limited number of antigen-encoding loci that are known to be under strong selection. We describe length variation at 12 microsatellite loci in 465 infections collected from 9 locations worldwide. These data reveal dramatic differences in parasite population structure in different locations. Strong linkage disequilibrium (LD) was observed in six of nine populations. Significant LD occurred in all locations with prevalence <1% and in only two of five of the populations from regions with higher transmission intensities. Where present, LD results largely from the presence of identical multilocus genotypes within populations, suggesting high levels of self-fertilization in populations with low levels of transmission. We also observed dramatic variation in diversity and geographical differentiation in different regions. Mean heterozygosities in South American countries (0.3-0.4) were less than half those observed in African locations (0. 76-0.8), with intermediate heterozygosities in the Southeast Asia/Pacific samples (0.51-0.65). Furthermore, variation was distributed among locations in South America (F:(ST) = 0.364) and within locations in Africa (F:(ST) = 0.007). The intraspecific patterns of diversity and genetic differentiation observed in P. falciparum are strikingly similar to those seen in interspecific comparisons of plants and animals with differing levels of outcrossing, suggesting that similar processes may be involved. The differences observed may also reflect the recent colonization of non-African populations from an African source, and the relative influences of epidemiology and population history are difficult to disentangle. These data reveal a range of population structures within a single pathogen species and suggest intimate links between patterns of epidemiology and genetic structure in this organism.

High-speed HPLC/MS/MS analysis of biological fluids: a practical review.

Over the course of the last three or four years, the need for increased throughput analytical methods in support of drug discovery operations has dramatically increased. Once a nicety, methods which can provide reliable quantitation of target analytes in biological fluids are now necessary to keep pace with biological screening. To date, the only successful approaches to direct high-speed quantitation of large numbers of diverse compounds are based on high-performance liquid chromatography/mass spectrometric (HPLC/MS/MS) techniques. Key challenges to this task include sample preparation, the automated development of methods and the rapid analysis of many samples. Accomplishing these tasks requires the use of generic methods, which can be applied across many structurally diverse compounds. In this review, the state-of-the-art in high-speed HPLC/MS/MS is reviewed with the aim of describing the most practical and effective approaches currently in use.

A new liquid chromatography/tandem mass spectrometric approach for the identification of class I major histocompatibility complex associated peptides that eliminates the need for bioassays.

Cell-surface class I major histocompatibility complex (MHC) molecules present processed self- and nonself-peptides to thymus-derived (T) lymphocytes, allowing the intracellular compartment of cells to be sampled in order to detect infection. Since the class I MHC-peptide complex plays a critical role in cell-mediated immunity, it is important to obtain sequence information on the MHC-associated peptides unique to infected cells as a prelude to the development of vaccines. Here, we outline and test an alternative strategy for identifying the proteins that are processed through the MHC pathway. This new strategy eliminates the necessity of developing and maintaining cytotoxic T lymphocyte (CTL) lines for peptide identification. In this new approach genome sequences from the infecting agent are scanned for stretches of amino acids that match a particular MHC binding motif. Molecular masses from these putative MHC-binding peptide sequences are calculated and compared to those found for peptides isolated from pathogen-infected host cells using liquid chromatography/mass spectrometry (LC/MS). Peptides with masses matching those in the database are then analyzed by tandem mass spectrometry (MS/MS) to determine their identity. Using this approach we were able to confirm the processing and presentation of two Trypanosoma cruzi proteins by the MHC class I pathway. These data suggest that a rigorous approach employing two-dimensional separations in conjunction with MS/MS and bioinformatics is a feasible means of identifying pathogen gene products of immunological interest when a CTL assay is unavailable or unsuccessful.

Optimization of a hydrophobic solid-phase extraction interface for matrix-assisted laser desorption/ionization.

Matrix-assisted laser desorption/ionization (MALDI) probe surfaces derivatized with octadecanethiol (C18) can be used as hydrophobic solid-phase extraction devices to isolate and desalt biopolymers directly on the probe surface. Using quantitative MALDI, it was possible to determine the approximate amount of peptide that bound to C18 surfaces and thus to calculate a surface density. It was determined that the amount of peptide bound at the probe surface was independent of the analyte concentration in the immersion solution (from high- to sub-ng ml-1 concentrations), but rather was dependent on the immersion time of the surface as it was exposed to the analyte. The capacity of C18-derivatized probes to bind biopolymers in fixed amounts frees the analyst from the necessity for adjusting analyte concentration through multiple step procedures such as serial dilution or vacuum drying. This time savings result in an overall increase in the efficiency of the MALDI technique.

On-probe solid-phase extraction/MALDI-MS using ion-pairing interactions for the cleanup of peptides and proteins.

Samples originating from biological sources often contain a complex mixture of inorganic salts, buffers, chaotropic agents, surfactants/detergents, preservatives, and other solubilizing agents. However, the presence of these contaminants virtually ensures the failure of any subsequent analysis of the sample by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Sample cleanup procedures, therefore, must be performed prior to MALDI-MS analysis. This paper reports a probe-surface derivatization method that greatly simplifies this sample preparation process. MALDI probes possessing self-assembled monolayers (SAMs) terminated with ionic functional groups can rapidly extract peptides/proteins via ionic interactions from < or = 1-microL volumes of sample solutions placed directly on their surface. We have found that MALDI probes modified in this manner are a practical solution for analyzing very small volumes of peptide/protein solutions contaminated with high levels of inorganic salts, buffers, detergents, chaotropic agents, and other solubilizing agents.

A desalting approach for MALDI-MS using on-probe hydrophobic self-assembled monolayers.

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.

New immobilization chemistry for probe affinity mass spectrometry.

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.

Probe-immobilized affinity chromatography/mass spectrometry.

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.