7th Annual Symposium on Clinical and Pharmaceutical Solutions through Analysis.

7th Annual Symposium on Clinical & Pharmaceutical Solutions through Analysis, Renaissance Shanghai Pudong Hotel, Shanghai, China, 20-23 April 2016 The 7th Annual Shanghai Symposium on Innovative Approaches to Reduce Attrition and Predict Clinical Outcomes (CPSA Shanghai 2016) was held on 20-23 April 2016 in Renaissance Shanghai Pudong Hotel, Shanghai, China. The meeting was featured with highly interactive events including diversified symposia, round table discussions, workshops, poster sessions and conference awards. There were over 220 participants from more than ten countries, with 61 oral presentations and 29 posters presented. In addition, the meeting included one preconference workshop and three joint sessions held with bioanalytical experts from local communities.

Effects of column and gradient lengths on peak capacity and peptide identification in nanoflow LC-MS/MS of complex proteomic samples.

Reversed-phase liquid chromatography is the most commonly used separation method for shotgun proteomics. Nanoflow chromatography has emerged as the preferred chromatography method for its increased sensitivity and separation. Despite its common use, there are a wide range of parameters and conditions used across research groups. These parameters have an effect on the quality of the chromatographic separation, which is critical to maximizing the number of peptide identifications and minimizing ion suppression. Here we examined the relationship between column lengths, gradient lengths, peptide identifications, and peptide peak capacity. We found that while longer column and gradient lengths generally increase peptide identifications, the degree of improvement is dependent on both parameters and is diminished at longer column and gradients. Peak capacity, in comparison, showed a more linear increase with column and gradient lengths. We discuss the discrepancy between these two results and some of the considerations that should be taken into account when deciding on the chromatographic conditions for a proteomics experiment.

Size-sorting combined with improved nanocapillary liquid chromatography-mass spectrometry for identification of intact proteins up to 80 kDa.

Despite the availability of ultra-high-resolution mass spectrometers, methods for separation and detection of intact proteins for proteome-scale analyses are still in a developmental phase. Here we report robust protocols for online LC-MS to drive high-throughput top-down proteomics in a fashion similar to that of bottom-up proteomics. Comparative work on protein standards showed that a polymeric stationary phase led to superior sensitivity over a silica-based medium in reversed-phase nanocapillary LC, with detection of proteins >50 kDa routinely accomplished in the linear ion trap of a hybrid Fourier transform mass spectrometer. Protein identification was enabled by nozzle-skimmer dissociation and detection of fragment ions with <10 ppm mass accuracy for highly specific database searching using tailored software. This overall approach led to identification of proteins up to 80 kDa, with 10-60 proteins identified in single LC-MS runs of samples from yeast and human cell lines prefractionated by their molecular mass using a gel-based sieving system.

Online nanoelectrospray/high-field asymmetric waveform ion mobility spectrometry as a potential tool for discovery pharmaceutical bioanalysis.

Nanoelectrospray ionization (nESI) coupled online with high-field asymmetric waveform ion mobility spectrometry (FAIMS) for small molecule analysis in a discovery pharmaceutical setting was examined. A conventional capillary pump, autosampler and nESI source were used to introduce samples directly into the FAIMS device. The FAIMS device was used to separate gas-phase ions on a timescale that was compatible with the mass spectrometer. The capability of the nESI-FAIMS combination to efficiently remove metabolite interferences from the parent drug, and reduce ion suppression effects, was demonstrated. On average, 85% of the signal intensity obtained from a neat sample was preserved in the extracted plasma samples. Standard curves were prepared for several compounds. Linearity was obtained over approximately 3 to 4 orders of magnitude. Comparison of results from nESI-FAIMS with those from conventional LC/MS for a mouse pharmacokinetic study yielded concentration values differing by no more than 30%.

Analysis of samples stored as individual plugs in a capillary by electrospray ionization mass spectrometry.

Droplets or plugs within multiphase microfluidic systems have rapidly gained interest as a way to manipulate samples and chemical reactions on the femtoliter to microliter scale. Chemical analysis of the plugs remains a challenge. We have discovered that nanoliter plugs of sample separated by air or oil can be analyzed by electrospray ionization mass spectrometry when pumped directly into a fused silica nanospray emitter tip. Using leucine-enkephalin in methanol and 1% acetic acid in water (50:50 v:v) as a model sample, we found carry-over between plugs was <0.1% and relative standard deviation of signal for a series of plugs was 3%. Detection limits were 1 nM. Sample analysis rates of 0.8 Hz were achieved by pumping 13 nL samples separated by 3 mm long air gaps in a 75 microm inner diameter tube. Analysis rates were limited by the scan time of the ion trap mass spectrometer. The system provides a robust, rapid, and information-rich method for chemical analysis of sample in segmented flow systems.

On-line 1D and 2D porous layer open tubular/LC-ESI-MS using 10-microm-i.d. poly(styrene-divinylbenzene) columns for ultrasensitive proteomic analysis.

Following on our recent work, on-line one-dimensional (1D) and two-dimensional (2D) porous layer open tubular/liquid chromatography-electrospray ionization-mass spectrometry (PLOT/LC-ESI-MS) platforms using 3.2 mx10 microm i.d. poly(styrene-divinylbenzene) (PS-DVB) PLOT columns have been developed to provide robust, high-performance, and ultrasensitive proteomic analysis. With the use of a PicoClear tee, the dead volume connection between a 50 microm i.d. PS-DVB monolithic micro-SPE column and the PLOT column was minimized. The micro-SPE/PLOT column assembly provided a separation performance similar to that obtained with direct injection onto the PLOT column at a mobile phase flow rate of 20 nL/min. The trace analysis potential of the platform was evaluated using an in-gel tryptic digest sample of a gel fraction (15-40 kDa) of a cervical cancer (SiHa) cell line. As an example of the sensitivity of the system, approximately 2.5 ng of protein in 2 microL of solution, an amount corresponding to 20 SiHa cells, was subjected to on-line micro-SPE-PLOT/LC-ESI-MS/MS analysis using a linear ion trap MS. A total of 237 peptides associated with 163 unique proteins were identified from a single analysis when using stringent criteria associated with a false positive rate of less than 1%. The number of identified peptides and proteins increased to 638 and 343, respectively, as the injection amount was raised to approximately 45 ng of protein, an amount corresponding to 350 SiHa cells. In comparison, only 338 peptides and 231 unique proteins were identified (false positive rate again less than 1%) from 750 ng of protein from the identical gel fraction, an amount corresponding to 6000 SiHa cells, using a typical 15 cmx75 microm i.d. packed capillary column. The greater sensitivity, higher recovery, and higher resolving power of the PLOT column resulted in the increased number of identifications from only approximately 5% of the injected sample amount. The resolving power of the micro-SPE/PLOT assembly was further extended by 2D chromatography via combination of the high-efficiency reversed-phase PLOT column with strong cation-exchange chromatography (SCX). As an example, 1071 peptides associated with 536 unique proteins were identified from 75 ng of protein from the same gel fraction, an amount corresponding to 600 cells, using five ion-exchange fractions in on-line 2D SCX-PLOT/LC-MS. The 2D system, implemented in an automated format, led to simple and robust operation for proteomic analysis. These promising results demonstrate the potential of the PLOT column for ultratrace analysis.

Ultra-low flow nanospray for the normalization of conventional liquid chromatography/mass spectrometry through equimolar response: standard-free quantitative estimation of metabolite levels in drug discovery.

Nanospray experiments were performed on an ensemble of drug molecules and their commonly known metabolites to compare performance with conventional electrospray ionization (ESI) and to evaluate equimolar response capabilities. Codeine, dextromethorphan, tolbutamide, phenobarbital, cocaine, and morphine were analyzed along with their well-known metabolites that were formed via hydroxylation, dealkylation, hydrolysis, and glucuronidation. Nanospray exhibited a distinct trend toward equimolar response when flow rate was reduced from 25 nL/min to less than 10 nL/min. A more uniform response between the parent drug and the corresponding metabolites was obtained at flow rates of 10 nL/min or lower. The largest discrepancy was within +/-50% for plasma samples. Nanospray was used as a calibrator for conventional ESI liquid chromatography/tandem mass spectrometry (LC/MS/MS) and normalization factors were applied to the quantitation of an acyl-glucuronide metabolite of a proprietary compound in rat plasma. A nanospray calibration method was developed with the standard curve of the parent drug to generate quantitative results for drug metabolites within +/-20% of that obtained with reference standards and conventional ESI. The nanospray method provides a practical solution for the quantitative estimation of drug metabolites in drug discovery when reference standards are not available.

Automated orthogonal control system for electrospray ionization.

Low-flow electrospray ionization is typically a purely electrostatic method, used without supporting sheath-gas nebulization. Complex spray morphology results from a large number of possible spray emission modes. Spray morphology may assume the optimal Taylor cone-jet spray mode under equilibrium conditions. When coupling to nanobore gradient elution chromatography, however, stability of the Taylor cone-jet spray mode is compromised by the gradient of mobile phase physiochemical properties. The common spray modes for aqueous/organic mobile phases were characterized using orthogonal (strobed illumination) transmitted light and (continuous illumination) scattered light imaging. Correlation of image sets from these complementary illumination methods provides the basis for spray mode identification using qualitative and quantitative image analysis. An automated feedback-controlled electrospray source was developed on a computer capable of controlling electrospray potential using an image-processing based algorithm for spray mode identification. The implementation of the feedback loop results in a system that is both self-starting and self-tuning for a specific spray mode or modes. Thus, changes in mobile phase composition and/or flow rate are compensated in real-time and the source is maintained in the cone-jet or pulsed cone-jet spray modes.

Miniaturized formats for efficient mass spectrometry-based proteomics and therapeutic development.

Off-line miniaturized "nano-spray" formats for electrospray ionization mass spectrometry (ESI-MS) enable the routine identification of femtomole quantities of protein or peptide. Even greater strides have been achieved using on-line miniaturized ESI-MS methods, such as nanobore LC-MS and CE-MS. On-line methods enable greater sensitivity (sub-attomole limit of detection), dynamic range, and throughput. In either off- or on-line methods for protein analysis, samples are typically isolated and digested enzymatically, with MS analysis of the peptide fragments, yielding 5-50% sequence coverage, in a "bottom-up" approach. Obtaining biologically relevant (structure/function) information (such as the localization of regions of error or post-transnational modifications) often demands 100% sequence coverage and this may be obtained by analyzing intact proteins by MS with a "top-down" methodology. Proteome wide success with top-down methods will require the development of novel miniaturized approaches for sample preparation along with new tools for bioinformatics. As these miniaturized formats continue to power proteomics applications, they will undoubtedly pollinate "cross-over" applications in LC-MS ranging from drug discovery to development. An example of metabolite identification using an order of magnitude less sample than usually required, with a concurrent order of magnitude increase in signal, illustrates the potential of miniaturized formats in lead characterization activities.