Pre-equilibration kinetic size-exclusion chromatography with mass spectrometry detection (peKSEC-MS) for label-free solution-based kinetic analysis of protein-small molecule interactions.

Here we introduce pre-equilibration kinetic size-exclusion chromatography with mass-spectrometry detection (peKSEC-MS), which is a label-free solution-based kinetic approach for characterizing non-covalent protein-small molecule interactions. In this method, a protein and a small molecule are mixed outside the column and incubated to approach equilibrium. The equilibrium mixture is then introduced into the SEC column to initiate the dissociation process by separating small molecules from the complex inside the column. A numerical model of a 1-dimensional separation was constructed to simulate mass chromatograms of the small molecule for varying rate constants of binding.

Kinetic size-exclusion chromatography with mass spectrometry detection: an approach for solution-based label-free kinetic analysis of protein-small molecule interactions.

Studying the kinetics of reversible protein-small molecule binding is a major challenge. The available approaches require that either the small molecule or the protein be modified by labeling or immobilization on a surface. Not only can such modifications be difficult to do but also they can drastically affect the kinetic parameters of the interaction. To solve this problem, we present kinetic size-exclusion chromatography with mass spectrometry detection (KSEC-MS), a solution-based label-free approach. KSEC-MS utilizes the ability of size-exclusion chromatography (SEC) to separate any small molecule from any protein-small molecule complex without immobilization and the ability of mass spectrometry (MS) to detect a small molecule without a label. The rate constants of complex formation and dissociation are deconvoluted from the temporal pattern of small molecule elution measured with MS at the exit from the SEC column. This work describes the concept of KSEC-MS and proves it in principle by measuring the rate constants of interaction between carbonic anhydrase and acetazolamide.

Multiplex enzyme assays and inhibitor screening by mass spectrometry.

Current methods for high-throughput screening (HTS) use a serial process to evaluate compounds as inhibitors toward a single therapeutic target, but as the demand to reduce screening time and cost continues to grow, one solution is the development of multiplex technology. In this communication, the multiplex assay capability of a mass spectrometry (MS)-based readout system is verified using a kinase and esterase reaction simultaneously. Furthermore, the MS-based readout is shown to be compatible with a typical HTS workflow by identifying and validating several new inhibitors for each enzyme from a small library of compounds. These data confirm that it is possible to monitor inhibition of multiple therapeutic targets with one pass through the compound repository, thus demonstrating the potential for MS-based methods to become a method of choice for HTS of isolated enzymes.

8-plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer's disease.

An 8-plex version of an isobaric reagent for the quantitation of proteins using shotgun methods is presented. The 8-plex version of the reagent relies on amine-labeling chemistry of peptides similar to 4-plex reagents. MS/MS reporter ions at 113, 114, 115, 116, 117, 118, 119, and 121 m/z are used to quantify protein expression. This technology which was first applied to a test mixture consisting of eight proteins and resulted in accurate quantitation, has the potential to increase throughput of analysis for quantitative shotgun proteomics experiments when compared to 2- and 4-plex methods. The technology was subsequently applied to a longitudinal study of cerebrospinal fluid (CSF) proteins from subjects undergoing intravenous Ig treatment for Alzheimer's disease. Results from this study identify a number of protein expression changes that occur in CSF after 3 and 6 months of treatment compared to a baseline and compared to a drug washout period. A visualization tool was developed for this dataset and is presented. The tool can aid in the identification of key peptides and measurements. One conclusion aided by the visualization tool is that there are differences in considering peptide-based observations versus protein-based observations from quantitative shotgun proteomics studies.

"One-pot" methylation in glycomics application: esterification of sialic acids and permanent charge construction.

A simple and rapid "one-pot" methylation method to esterify sialic acids and construct a permanent charge was developed for N-linked glycan analysis, which combined complete nonspecific proteolytic digestion and methylation. A mixture of Asn-glycans prepared from Pronase E digestion of the glycoprotein was passed through a cation-exchange column to convert carboxylic acids to the Na+ form before being methylated with methyl iodide. Derivatives could be easily purified with a hydrophilic affinity chromatography cartridge. Mass spectrometry analysis was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and MALDI-TOF/TOF. The mass spectrometric data indicated that carboxylic acids were methylated in addition to the formation of a quaternary ammonium in the amino group of asparagine residues. Three model glycoproteins, including ribonuclease B, ovalbumin, and transferrin, were employed to demonstrate the merits of this technique. Results showed that the stabilization of sialic acid was achieved in addition to the formation of a permanent charge. Compared to the analysis of underivatized N-glycans, detection sensitivity improved approximately 10-fold. The new technique was further evaluated with glycan profiling of serum transferrin and proved to be a sensitive method for the characterizing protein glycosylation.

Characterization of Enterobacteria using MALDI-TOF mass spectrometry.

A method is proposed for the rapid classification of Gram-negative Enterobacteria using on-slide solubilization and trypsin digestion of proteins, followed by MALDI-TOF MS analysis. Peptides were identified from tryptic digests using microsequencing by tandem mass spectrometry and database searches. Proteins from the outer membrane family (OMP) were consistently identified in the Enterobacteria Escherichia coli, Enterobacter cloacae, Erwinia herbicola, and Salmonella typhimurium. Database searches indicate that these OMP peptides observed are unique to the Enterobacteria order.

Tn10 transposase mutants with altered transpososome unfolding properties are defective in hairpin formation.

Transposition reactions take place in the context of higher-order protein-DNA complexes called transpososomes. In the Tn10 transpososome, IHF binding to an "outside end" creates a bend in the DNA that allows the transposase protein to contact the end at two different sites, the terminal and subterminal binding sites. Presumably this helps to stabilize the transposase-end interaction. However, the DNA loop that is formed must be unfolded at a later stage in order for the transposon to integrate into other DNA molecules. It has been proposed that transpososome unfolding also plays a role in transposon excision. To investigate this possibility further, we have isolated and characterized transposase mutants with altered transpososome unfolding properties. Two such mutants were identified, R182A and R184A. Both mutants fail to carry out hairpin formation, an intermediate step in transposon excision, specifically with outside end-containing substrates. These results support the idea that transpososome unfolding and excision are linked. Also, based on the importance of residues R182 and R184 in transpososome unfolding, we propose a new model for the Tn10 transpososome, wherein both DNA ends of the transpososome make subterminal contacts with transposase.

Rapid characterization of Bacillus spores targeting species-unique peptides produced with an atmospheric pressure matrix-assisted laser desorption/ionization source.

New and improved strategies are eagerly sought for the rapid identification of microorganisms, particularly in mixtures. Mass spectrometry remains a powerful tool for this purpose. Small acid-soluble proteins (SASPs), which are relatively abundant in Bacillus spores, represent potential biomarkers for species characterization. Despite sharing extensive sequence homology, these proteins differ sufficiently in sequence for discrimination between species. This work focuses on the differences in sequence between SASPs from various Bacillus species. Compilation of SASP sequences from protein database searches, followed by in silico trypsin digestion and analysis of the resulting fragments, identified several species-specific peptides that could be targeted for analysis using mass spectrometry. This strategy was tested and found to be successful in the characterization of Bacillus spores both from individual species and in mixtures. Analysis was performed using an ion trap mass spectrometer with an atmospheric pressure MALDI source. This instrumentation offers the advantage of increased speed of analysis and accurate precursor ion selection for tandem mass spectrometric analysis compared with vacuum matrix-assisted laser desorption/ionization and time-of-flight instruments. The identification and targeting of species-specific peptides using this type of instrumentation offers a rapid, efficient strategy for the identification of Bacillus spores and can potentially be applied to different microorganisms.

Enhancement and rescue of target capture in Tn10 transposition by site-specific modifications in target DNA.

The bacterial transposon Tn10 inserts preferentially into specific target sequences. This insertion specificity appears to be linked to the ability of target sites to adopt symmetrically positioned DNA bends after binding the transposition machinery. Target DNA bending is thought to permit the transposase protein to make additional contacts with the target DNA, thereby stabilizing the target complex so that the joining of transposon and target DNA sequences can occur efficiently. In the current work, we have asked whether the introduction of a discontinuity in a target DNA strand, a modification that is expected to make it easier for a DNA molecule to bend, can enhance or rescue target capture under otherwise suboptimal reaction conditions. We show that either a nick or a missing phosphate specifically at the site of reaction chemistry increases the ability of various target DNAs to form the target capture complex. The result suggests that the bends in the target DNA are highly localized and include the scissile phosphates. This raises the possibility that strand transfer is mechanistically linked to target capture. We have also identified specific residues in the target DNA and in transposase that appear to play an important role in target DNA bending.

Target DNA bending is an important specificity determinant in target site selection in Tn10 transposition.

The bacterial transposon Tn10 inserts preferentially into specific DNA sequences. DNA footprinting and interference studies have revealed that the Tn10-encoded transposase protein contacts a large stretch of target DNA ( approximately 24 bp) and that the target DNA structure is deformed upon incorporation into the transpososome. Target DNA deformation might contribute significantly to target site selection and thus it is of interest to further define the nature of this deformation. Circular permutation analysis was used to demonstrate that the target DNA is bent upon its incorporation into the transpososome. Two lines of evidence are presented that target DNA bending is an important event in target site selection. First, we demonstrate a correlation between increased target site usage and an increased level of target DNA bending. Second, transposase mutants with relaxed target specificity are shown to cause increased target DNA bending relative to wild-type transposase. This latter observation provides new insight into how relaxed specificity may be achieved. We also show that Ca(2+) facilitates target capture by stabilizing transposase interactions with sequences immediately flanking the insertion site. Ca(2+) could, in theory, exert this effect by stabilizing bends in the target DNA.