Development and characterization of a novel plug and play liquid chromatography-mass spectrometry (LC-MS) source that automates connections between the capillary trap, column, and emitter.

We report the development and characterization of a novel, vendor-neutral ultra-high pressure-compatible (~10,000 p.s.i.) LC-MS source. This device is the first to make automated connections with user-packed capillary traps, columns, and capillary emitters. The source uses plastic rectangular inserts (referred to here as cartridges) where individual components (i.e. trap, column, or emitter) can be exchanged independent of one another in a plug and play manner. Automated robotic connections are made between the three cartridges using linear translation powered by stepper motors to axially compress each cartridge by applying a well controlled constant compression force to each commercial LC fitting. The user has the versatility to tailor the separation (e.g. the length of the column, type of stationary phase, and mode of separation) to the experimental design of interest in a cost-effective manner. The source is described in detail, and several experiments are performed to evaluate the robustness of both the system and the exchange of the individual trap and emitter cartridges. The standard deviation in the retention time of four targeted peptides from a standard digest interlaced with a soluble Caenorhabditis elegans lysate ranged between 3.1 and 5.3 s over 3 days of analyses. Exchange of the emitter cartridge was found to have an insignificant effect on the abundance of various peptides. In addition, the trap cartridge can be replaced with minimal effects on retention time (<20 s).

Regulated phosphorylation of budding yeast's essential myosin V heavy chain, Myo2p.

The tail of the yeast myosin V encoded by Myo2p is known to bind several receptors for cargo delivery along polarized actin cables. However, it is not known how Myo2p activity is regulated or how it selects between cargoes. Here we show that Myo2p is reversibly phosphorylated in vivo. A short peptide at the N-terminal end of the cargo-binding domain contains three residues contributing to single or doubly phosphorylated species. We confirm that the tail consists of two proteolytically resistant subdomains and identify a functionally important region N-terminal to subdomain 1 that includes the phosphorylation sites. Mutagenesis of the phosphorylation sites to alanine abolished a mobility shift diagnostic of phosphorylation, whereas mutagenesis to glutamic acid produced the shift and the formation of an additional phosphorylated species. These substitutions did not affect overall cell growth. However, one of the sites is predicted to be a substrate of cAMP-dependent protein kinase (PKA), and yeast expressing Myo2p with alanine substitutions is resistant to otherwise lethal overexpression of PKA, whereas the glutamic acid mutant is supersensitive to overexpression of PKA. These results suggest that in yeast, Myo2p is subject to phosphoregulation involving a PKA-related signaling pathway.

Automated nano-electrospray mass spectrometry for protein-ligand screening by noncovalent interaction applied to human H-FABP and A-FABP.

A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Futhermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by HTS.

Chip-based nanoelectrospray mass spectrometry for protein characterization.

In the last several years, significant progress has been made in the development of microfluidic-based analytical technologies for proteomic and drug discovery applications. Chip-based nanoelectrospray coupled to a mass spectrometer detector is one of the recently developed analytical microscale technologies. This technology offers unique advantages for automated nanoelectrospray including reduced sample consumption, improved detection sensitivity and enhanced data quality for proteomic studies. This review presents an overview and introduction of recent developments in chip devices coupled to electrospray mass spectrometers including the development of the automated nanoelectrospray ionization chip device for protein characterization. Applications using automated chip-based nanoelectrospray ionization technology in proteomic and bioanalytical studies are also extensively reviewed in the fields of high-throughput protein identification, protein post-translational modification studies, top-down proteomics, biomarker screening by pattern recognition, noncovalent protein-ligand binding for drug discovery and lipid analysis. Additionally, future trends in chip-based nanoelectrospray technology are discussed.

Detection of single nucleotide polymorphisms using electrospray ionization mass spectrometry: validation of a one-well assay and quantitative pooling studies.

Single nucleotide polymorphisms (SNPs) are currently being mapped and databased at a remarkable pace, providing a viable means for understanding disease susceptibility, differential drug response and human evolution. Consequently, there is an increasing demand for SNP genotyping technologies that are simple, rapid, cost effective and readily amenable to automation for high-throughput analyses. In this study, we improved the Survivor Assay, a SNP detection method based on electrospray ionization mass spectrometry (ESI-MS), with several developments. One improvement is the development of a one-well assay, requiring no off-line purification of the polymerase chain reaction product, achieved by simple addition of reagent solution into a single well. Another is the on-line separation of magnesium and dideoxynucleotides using an in-house made monolithic metal chelating column, eliminating any off-line sample preparation prior to mass spectrometric analysis. Here the Survivor Assay is extended from a proof-of-principle concept to a validated method by genotyping six SNPs from five different regions of human genomic DNA in 55 individual samples with 100% accuracy. This improved Survivor Assay eliminates the tedious and time-consuming steps of sample preparation, minimizes sample handing and offers a high-throughput analysis of SNPs by ESI-MS. The current combined preparation and analysis time is 2 min per sample. The simplicity of this method has potential for full automation and parallel chromatography and, thus, reduced analysis time. In addition, we have adapted the Survivor Assay for quantitative SNP analysis in pooled DNA samples. The capabilities and sensitivity of this approach were evaluated. We demonstrate that an allele occurring at a frequency of 2% can consistently be quantitated.

Determination of SCH 211803 by nanoelectrospray infusion mass spectrometry: evaluation of matrix effect and comparison with liquid chromatography-tandem mass spectrometry.

A high throughput assay for SCH 211803, an M2 muscarinic receptor antagonist in human plasma using nanoelectrospray infusion tandem mass spectrometry is described. Sample processing consisted of protein precipitation followed by solid phase extraction using octadecasilyl resin-filled pipette tips on a liquid handling robotic system. The sample extracts were infused directly to the mass spectrometer using a nanoelectrospray interface in a silicon chip format. SCH 211803 was quantified in plasma over the concentration range of 1-1000 ng/mL. In comparison with a liquid chromatography-tandem mass spectrometry assay, the nanoelectrospray method has comparable accuracy, precision and limit of quantitation, with a nine-fold improvement in sample throughput. Using the nanoelectrospray assay, ion suppression was evaluated and found to be 15%. This represented a four-fold reduction in matrix suppression when compared to a conventional electrospray source operating in the flow injection analysis mode at a flow rate common for LC-MS/MS analysis.

Ultralow-volume fraction collection from NanoLC columns for mass spectrometric analysis of protein phosphorylation and glycosylation.

An ultralow volume fraction collection system referred to as nano fraction analysis chip technology (nanoFACT) is reported. The system collects 25-2500-nL fractions from 75-microm nanoLC columns into pipet tips at a user-defined, timed interval, typically one fraction every 15-120 s. Following collection, the fractions in the tip dry down naturally on their own in such a way as to create a concentrated band at the very end of the interior of the pipet tip. The fractions are then reconstituted directly in the pipet tips in approximately 250 nL of solvent prior to analysis. Because the chromatography and reconstitution solvent are independent, the reconstitution solvent can be selected to maximize ionization efficiency without compromising chromatography. In the infusion analysis of the nanoLC fractions, a low-flow electrospray chip is used which consists of 400 nozzles, each with an inner diameter of 2.5 microm and yielding flow rates of approximately 20 nL/min. Therefore, when reconstituted in 250 nL, each nanoLC fraction can be analyzed for over 10 min. This increase in analysis time allows for signal averaging, resulting in higher data quality, collision energy optimization, slower scanning techniques to be used, such as neutral loss and precursor ion scanning, higher resolution scans on FTMS instruments, and improved peptide quantitation. Furthermore, the nanoLC fractions could be archived in the pipet tips for analysis at a later date. Here, the advantages of nanoFACT are shown for phosphorylation analysis using bovine fetuin and glycosylation analysis using bovine ribonuclease B (RNase B). In the phosphorylation analysis, a comparison between conventional nanoLC and a nanoFACT analysis was performed. An MS/MS spectrum of a triply phosphorylated peptide, 313-HTFSGVApSVEpSpSSGEAFHVGK-333 could only be obtained using nanoFACT, not with nanoLC. Furthermore, spectral quality for the nanoFACT analysis was significantly improved over nanoLC. This was determined by comparing the number of diagnostic ions between the nanoFACT and nanoLC spectra, and it was found that the nanoFACT spectra contained a 19% or greater number of diagnostic ions for nonphosphorylated peptides and 55% or greater for phosphorylated peptides. For the glycosylation analysis, the glycosylation site of RNase B was fully characterized using 100 fmol of tryptic digest on a three-dimensional ion trap mass spectrometer.

Characterization of a fully automated nanoelectrospray system with mass spectrometric detection for proteomic analyses.

Although nanoelectrospray offers low sample consumption and improved detection limits for proteomic studies, it is currently a low throughput technique because of its tedious single-sprayer alignment procedures. Here, a fully automated nanoelectrospray system for proteomic analyses with mass spectrometric detection is described and characterized. This infusion system aspirates samples from a 96-well plate using disposable pipette tips and then delivers samples to an ESI Chip. This chip is a fully integrated monolithic device consisting of a 10 x 10 array of nozzles. The automated nanoelectrospray system is easily controlled with software that allows the user simply to select the number of samples to analyze, the volume of sample to aspirate, and the spray voltage and head pressure to apply. The system offers all the advantages of conventional nanoelectrospray plus automated, high throughput analyses with no carryover. The high degree of reproducibility and lack of carryover of the system are demonstrated here using a Micromass LCT time-of-flight mass spectrometer with the infusion of six tryptic digests through all 100 nozzles of a chip. The effects of ammonium acetate and sodium dodecyl sulfate are discussed, as well as the system's ability to spray a variety of different solvents. The spray stability of whole cytochrome c in 99.9% water with 0.1% acetic acid over a 15-min period was determined to be 5.06%. Using a Thermo Finnigan LCQ Deca ion trap and SEQUEST search software, 2 fmol/microL myoglobin and 1 fmol/microL cytochrome c digests were unambiguously identified via infusion analyses. Finally, protein spots excised from two-dimensional gels of yeast and E. coli crude cell extracts were identified with the fully automated nanoelectrospray system coupled to an LCQ.

Quantitative determination of noncovalent binding interactions using automated nanoelectrospray mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) has proven to be an extremely powerful tool for studying biomolecular structures and noncovalent interactions. Here we report a method using a fully automated, chip-based nanoESI-MS system to determine the dissociation constants (Kd) for the complexes of two different proteins with their ligands. The automated nanoelectrospray system, consisting of the NanoMate and ESI chip, serves functionally as a combination of autosampler and nanoelectrospray ionization source. This system provides all the advantages of conventional nanoelectrospray plus automated, high-throughput analyses without carryover. The automated nanoESI system was used to investigate quantitative noncovalent interactions between ribonuclease A (RNase A) and cytidylic acid ligands (2'-CMP, CTP), a well-characterized model protein-ligand complex, and between an inactive endocellulase mutant (Thermobifida fusca Cel6A D117Acd) and four oligosaccharide ligands (cellotriose, cellotetraose, cellopentaose, cellohexaose). Both titration and competitive binding approaches were performed prior to automated nanoESI-MS analysis with a Q-TOF mass spectrometer. Dissociation constants for each complex were calculated from the sum of ion peak areas of free and complexed proteins during the titration and competition experiments. The measured Kd values for the RNase A-CMP and Cel6A D117Acd-G3 complexes were found to be in excellent agreement with the available published values obtained by standard spectroscopic titration techniques. To our knowledge, this is the first report of using an ESI-MS approach to study the interactions between a cellulase and oligosaccharides. The results provide new insights for understanding the nature of cellulase-cellulose interactions.

Automated chip-based nanoelectrospray-mass spectrometry for rapid identification of proteins separated by two-dimensional gel electrophoresis.

We report a method using a fully automated chip-based nanoelectrospray system for two-dimensional (2-D) gel sample analyses with mass spectrometric detection. The automated nanoelectrospray system, consisting of the NanoMate and electrospray ionization (ESI) chip, serves as both an autosampler and nanoESI source. This infusion system aspirates samples from a 96-well plate using disposable pipette tips and then delivers these samples sequentially to an ESI chip. This chip is a fully integrated monolithic device consisting of a 10x10 array of nozzles. The automated nanoelectrospray system is easily controlled through software, permitting the user to select the number of samples to be analyzed, the volume of sample to aspirate, the spray voltage, and analysis time. The system offers all the advantages of conventional nanoelectrospray plus automated, high-throughput analyses without analyte carryover. The system was used for a protein identification study of 2-D gel spots of both Escherichia coli and yeast crude cell extracts. The identification of 50 spots from E. coli crude cell extract and 27 spots from yeast extract is presented, demonstrating the powerful combination of the automated nanoESI system, the Thermo Finnigan LCQ Deca ion-trap mass spectrometer, and SEQUEST search software. In addition, the effects of silver staining and colloidal Coomassie blue staining of 2-D gel spots on the detection sensitivity and protein sequence coverage are compared and discussed. Furthermore, the comparison results using the multiwell microscale preparation kit versus manual extraction for in-gel samples are presented.

Quantitation of midazolam in human plasma by automated chip-based infusion nanoelectrospray tandem mass spectrometry.

An automated chip-based infusion nanoelectrospray ionization (nanoESI) platform was used to demonstrate reproducible quantitation of drug molecules from biological matrices. Three sample preparation strategies were explored including protein precipitation of plasma with acetonitrile, de-salting of the plasma, and a combination of protein precipitation with subsequent de-salting of the dried and reconstituted extract. The best results were obtained when fortified human plasma samples containing midazolam were precipitated with acetonitrile containing alprazolam as the internal standard (IS). The supernatant was concentrated to dryness, reconstituted in aqueous acid, and de-salted by automated reversed-phase solid-phase extraction (SPE) prior to infusion nanoESI-MS/MS. Analyses employed a triple quadrupole mass spectrometer operated in selected reaction monitoring (SRM) mode. Each sample was infused for approximately 10 s and the resulting ion current profiles were integrated. Area ratios were used for regression analysis of standard samples (1.5-500 ng/mL). Quality control samples (3, 250, and 400 ng/mL) in five replicates from three different analysis days demonstrated intra-assay precision (< or =16%), inter-assay precision (< or =5%), and overall accuracy (+/-9% deviation). Infusion reproducibility of the assay was established by analyzing extracts after storage for 24 h at ambient temperature. Control plasma samples from six different sources probed the potential utility of this technique for the analysis of clinical samples. At the lower limit of quantitation (LLQ), variability and mean overall accuracy were < or =13% CV and +/-3% deviation, respectively, while at the upper limit of quantitation (ULQ) variability and mean overall accuracy were < or =9% CV and +/-9% deviation, respectively. Inter-chip variability was established by determining standard sample extracts across five different chips (< or =12% CV). Throughput for the assay was 55 s per sample, although this time may be shortened to 40 s per sample with recent improvements in the automated nanoESI system. No contamination or carryover was observed using this promising automated nanoESI-MS/MS platform.

A fully automated nanoelectrospray tandem mass spectrometric method for analysis of Caco-2 samples.

Caco-2 cells offer a means to rapidly screen permeability of drug candidates, allowing pharmaceutical companies to eliminate candidates unable to cross the intestinal barrier early in the discovery process. This screening process is typically performed by conventional liquid chromatography/tandem mass spectrometry (LC/MS/MS), which can require time-consuming method development. An alternative to LC/MS/MS, automated nanoelectrospray tandem mass spectrometry (nanoESI-MS/MS), is introduced. This novel approach requires an off-line ZipTip desalting step followed by automated nanoESI-MS/MS, using the NanoMate 100 and ESI Chip. In addition to reduced method development time, automated nanoESI-MS/MS also offers no carry-over between samples, low sample consumption, and ease-of-use as compared with conventional pulled-capillary nanoelectrospray. Furthermore, the infusion system described has the potential to be high-throughput. A comparison of Caco-2 samples analyzed both by LC/MS/MS and by automated nanoESI-MS/MS is presented. The permeability and recovery data of the two compounds analyzed in this study obtained from conventional LC/MS/MS and by automated nanoESI-MS/MS were in excellent agreement.