A combined high-throughput and high-content platform for unified on-chip synthesis, characterization and biological screening.

Acceleration and unification of drug discovery is important to reduce the effort and cost of new drug development. Diverse chemical and biological conditions, specialized infrastructure and incompatibility between existing analytical methods with high-throughput, nanoliter scale chemistry make the whole drug discovery process lengthy and expensive. Here, we demonstrate a chemBIOS platform combining on-chip chemical synthesis, characterization and biological screening. We developed a dendrimer-based surface patterning that enables the generation of high-density nanodroplet arrays for both organic and aqueous liquids. Each droplet (among > 50,000 droplets per plate) functions as an individual, spatially separated nanovessel, that can be used for solution-based synthesis or analytical assays. An additional indium-tin oxide coating enables ultra-fast on-chip detection down to the attomole per droplet by matrix-assisted laser desorption/ionization mass spectrometry. The excellent optical properties of the chemBIOS platform allow for on-chip characterization and in-situ reaction monitoring in the ultraviolet, visible (on-chip UV-Vis spectroscopy and optical microscopy) and infrared (on-chip IR spectroscopy) regions. The platform is compatible with various cell-biological screenings, which opens new avenues in the fields of high-throughput synthesis and drug discovery.

Soft cluster-induced desorption/ionization mass spectrometry: How soft is soft?

Desorption/ionization induced by neutral clusters (DINeC) is used as an ultrasoft desorption/ionization method for the analysis of fragile biomolecules by means of mass spectrometry (MS). As a test molecule, the glycopeptide vancomycin was measured with DINeC-MS, and resulting mass spectra were compared to the results obtained with electrospray ionization (ESI), matrix assisted laser desorption ionization, and time-of-flight secondary ion MS. Of the desorption-based techniques, DINeC spectra show the lowest abundance of fragments comparable to ESI spectra. The soft desorption nature of DINeC was further demonstrated when applied to MS analysis of teicoplanin.

Evaluation of Distance Metrics and Spatial Autocorrelation in Uniform Manifold Approximation and Projection Applied to Mass Spectrometry Imaging Data.

In this work, uniform manifold approximation and projection (UMAP) is applied for nonlinear dimensionality reduction and visualization of mass spectrometry imaging (MSI) data. We evaluate the performance of the UMAP algorithm on MSI data sets acquired in mouse pancreas and human lymphoma samples and compare it to those of principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), and the Barnes-Hut (BH) approximation of t-SNE. Furthermore, we compare different distance metrics in (BH) t-SNE and UMAP and propose the use of spatial autocorrelation as a means of comparing the resulting low-dimensional embeddings. The results indicate that UMAP is competitive with t-SNE in terms of visualization and is well-suited for the dimensionality reduction of large (>100 000 pixels) MSI data sets. With an almost fourfold decrease in runtime, it is more scalable in comparison with the current state-of-the-art: t-SNE or the Barnes-Hut approximation of t-SNE. In what seems to be the first application of UMAP to MSI data, we assess the value of applying alternative distance metrics, such as the correlation, cosine, and the Chebyshev metric, in contrast to the traditionally used Euclidean distance metric. Furthermore, we propose "histomatch" as an additional custom distance metric for the analysis of MSI data.

The downfall of TBA-354 - a possible explanation for its neurotoxicity via mass spectrometric imaging.

1. TBA-354 was a promising antitubercular compound with activity against both replicating and static Mycobacterium tuberculosis (M.tb), making it the focal point of many clinical trials conducted by the TB Alliance. However, findings from these trials have shown that TBA-354 results in mild signs of reversible neurotoxicity; this left the TB Alliance with no other choice but to stop the research. 2. In this study, mass spectrometric methods were used to evaluate the pharmacokinetics and spatial distribution of TBA-354 in the brain using a validated liquid chromatography tandem-mass spectrometry (LCMS/MS) and mass spectrometric imaging (MSI), respectively. Healthy female Sprague-Dawley rats received intraperitoneal (i.p.) doses of TBA-354 (20 mg/kg bw). 3. The concentrationtime profiles showed a gradual absorption and tissue penetration of TBA-354 reaching the Cmax at 6 h post dose, followed by a rapid elimination. MSI analysis showed a time-dependent drug distribution, with highest drug concentration mainly in the neocortical regions of the brain. 4. The distribution of TBA-354 provides a possible explanation for the motor dysfunction observed in clinical trials. These results prove the importance of MSI as a potential tool in preclinical evaluations of suspected neurotoxic compounds.

Low Abundant N-linked Glycosylation in Hen Egg White Lysozyme Is Localized at Nonconsensus Sites.

Although wild-type hen egg white lysozyme (HEL) is lacking the consensus sequence motif NX(S/T), in 1995 Trudel et al. (Biochem. Cell Biol. 1995, 73, 307-309) proposed the existence of a low abundant N-glycosylated form of HEL; however, the identity of active glycosylation sites in HEL remained a matter of speculation. For the first time since Trudel's initial work, we report here a comprehensive characterization by means of mass spectrometry of N-glycosylation in wild-type HEL. Our analytical approach comprised ZIC-HILIC enrichment of N-glycopeptides from HEL trypsin digest, deglycosylation by (18)O/PNGase F as well as by various endoglycosidases, and LC-MS/MS analysis of both intact and deglycosylated N-glycopeptides engaging multiple techniques of ionization and fragmentation. A novel data interpretation workflow based on MS/MS spectra classification and glycan database searching enabled the straightforward identification of the asparagine-rich N-glycopeptide [34-45] FESNFNTQATNR and allowed for compositional profiling of its modifying N-glycans. The overall heterogeneity profile of N-glycans in HEL comprised at least 26 different compositions. Results obtained from deglycosylation experiments provided clear evidence of asparagine residues N44 and N39 representing active glycosylation sites in HEL. Both of these sites do not fall into any known N-glycosylation-specific sequence motif but are localized in rarely observed nonconsensus sequons (NXN, NXQ).

The SMN interactome includes Myb-binding protein 1a.

Understanding networks of interacting proteins is a major goal in cell biology. The survival of motor neurons protein (SMN) interacts, directly or indirectly, with a large number of other proteins and reduced levels of SMN cause the inherited disorder spinal muscular atrophy (SMA). Some SMN interactions are stable and stoichiometric, such as those with gemins, while others are expected to be transient and substoichiometric, such as the functional interaction of SMN with coilin in Cajal bodies. This study set out to determine whether novel components of the extensive SMN interactome can be identified by a proteomic approach. SMN complexes were immuno-precipitated from HeLa nuclear extracts, using anti-SMN monoclonal antibody attached to magnetic beads, digested with trypsin, separated by capillary-liquid chromatography and analyzed by MALDI TOF/TOF mass spectrometry. One-hundred and one proteins were detected with a p value of <0.05, SMN, gemins and U snRNPs being the dominant "hits". Sixty-nine of these were rejected after MALDI analysis of two control pull-downs using antibodies against unrelated nuclear proteins. The proteins found only in anti-SMN pulldowns were either known SMN partners, and/or contained dimethylated RG domains involved in direct interaction with the SMN tudor domain, or they were known binding partners of such direct SMN interactors. Myb-binding protein 1a, identified as a novel candidate, is a mainly nucleolar protein of unknown function but it partially colocalized with SMN in Cajal bodies in HeLa cell nucleoplasm and, like SMN, was reduced in cells from an SMA patient.

Analysis of glycoproteins in human serum by means of glycospecific magnetic bead separation and LC-MALDI-TOF/TOF analysis with automated glycopeptide detection.

Comprehensive proteomic analyses require efficient and selective pre-fractionation to facilitate analysis of post-translationally modified peptides and proteins, and automated analysis workflows enabling the detection, identification, and structural characterization of the corresponding peptide modifications. Human serum contains a high number of glycoproteins, comprising several orders of magnitude in concentration. Thereby, isolation and subsequent identification of low-abundant glycoproteins from serum is a challenging task. selective capturing of glycopeptides and -proteins was attained by means of magnetic particles specifically functionalized with lectins or boronic acids that bind to various structural motifs. Human serum was incubated with differentially functionalized magnetic micro-particles (lectins or boronic acids), and isolated proteins were digested with trypsin. Subsequently, the resulting complex mixture of peptides and glycopeptides was subjected to LC-MALDI analysis and database searching. In parallel, a second magnetic bead capturing was performed on the peptide level to separate and analyze by LC-MALDI intact glycopeptides, both peptide sequence and glycan structure. Detection of glycopeptides was achieved by means of a software algorithm that allows extraction and characterization of potential glycopeptide candidates from large LC-MALDI-MS/MS data sets, based on N-glycopeptide-specific fragmentation patterns and characteristic fragment mass peaks, respectively. By means of fast and simple glycospecific capturing applied in conjunction with extensive LC-MALDI-MS/MS analysis and novel data analysis tools, a high number of low-abundant proteins were identified, comprising known or predicted glycosylation sites. According to the specific binding preferences of the different types of beads, complementary results were obtained from the experiments using either magnetic ConA-, LCA-, WGA-, and boronic acid beads, respectively.

N-terminal H3/D3-acetylation for improved high-throughput peptide sequencing by matrix-assisted laser desorption/ionization mass spectrometry with a time-of-flight/time-of-flight analyzer.

A novel method for peptide sequencing by matrix-assisted laser desorption/ionization mass spectrometry with a time-of-flight/time-of-flight analyzer (MALDI-TOF/TOF) is presented. A stable isotope label introduced in the peptide N-terminus by derivatization, using a 1:1 mixture of acetic anhydride and deuterated acetic anhydride, allows for easy and unambiguous identification of ions belonging either to the N- or the C-terminal ion series in the product ion spectrum, making sequence assignment significantly simplified. The good performance of this technique was shown by successful sequencing of the contents of several peptide maps. A similar approach was recently applied to nanoelectrospray ionization (nanoESI) and nano-liquid chromatography/tandem mass spectrometry (LC/MS/MS). The MALDI-TOF/TOF technique allows for fast, direct sequencing of modified peptides in proteomics samples, and is complementary to the nanoESI and nanoLC/MS/MS approaches.

Molecular classification of liver cirrhosis in a rat model by proteomics and bioinformatics.

Liver cirrhosis is a worldwide health problem. Reliable, noninvasive methods for early detection of liver cirrhosis are not available. Using a three-step approach, we classified sera from rats with liver cirrhosis following different treatment insults. The approach consisted of: (i) protein profiling using surface-enhanced laser desorption/ionization (SELDI) technology; (ii) selection of a statistically significant serum biomarker set using machine learning algorithms; and (iii) identification of selected serum biomarkers by peptide sequencing. We generated serum protein profiles from three groups of rats: (i) normal (n=8), (ii) thioacetamide-induced liver cirrhosis (n=22), and (iii) bile duct ligation-induced liver fibrosis (n=5) using a weak cation exchanger surface. Profiling data were further analyzed by a recursive support vector machine algorithm to select a panel of statistically significant biomarkers for class prediction. Sensitivity and specificity of classification using the selected protein marker set were higher than 92%. A consistently down-regulated 3495 Da protein in cirrhosis samples was one of the selected significant biomarkers. This 3495 Da protein was purified on-chip and trypsin digested. Further structural characterization of this biomarkers candidate was done by using cross-platform matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting (PMF) and matrix-assisted laser desorption/ionization time of flight/time of flight (MALDI-TOF/TOF) tandem mass spectrometry (MS/MS). Combined data from PMF and MS/MS spectra of two tryptic peptides suggested that this 3495 Da protein shared homology to a histidine-rich glycoprotein. These results demonstrated a novel approach to discovery of new biomarkers for early detection of liver cirrhosis and classification of liver diseases.

Comparison of laser-induced dissociation and high-energy collision-induced dissociation using matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) for peptide and protein identification.

The fragmentation of peptides under laser-induced dissociation (LID) as well as high-energy collision-induced dissociation (CID) conditions has been investigated. The effect of the different fragmentation mechanisms on the formation of specific fragment ion types and the usability of the resulting spectra, e.g. for high-throughput protein identification, has been evaluated. Also, basic investigations on the influence of the matrix, as well as laser fluence, on the fragment ion formation and the consequences in the spectral appearance are discussed. The preconditions for obtaining 'pure' CID spectra on matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) instruments are evaluated and discussed as well as the differences between LID and CID in the resulting fragment ion types. While containing a wealth of information due to additional fragment ions in comparison with LID, CID spectra are significantly more complex than LID spectra and, due to different fragmentation patterns, the CID spectra are of limited use for protein identification, even under optimized parameter settings, due to significantly lower scores for the individual spectra. Conditions for optimal results regarding protein identification using MALDI-TOF/TOF instruments have been evaluated. For database searches using tandem mass spectrometric data, the use of LID as fragmentation technique in combination with parameter settings supporting the use of internal fragment ions turned out to yield the optimal results.

Trace determination of priority pesticides in water by means of high-speed on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry using turbulent-flow chromatography columns for enrichment and a short monolithic column for fast liquid chromatographic separation.

An integrated on-line SPE-HPLC-MS/MS system has been developed for the rapid analysis of various trace level priority pesticides in surface and drinking water. Eleven pesticides were included in this study, with various phenylureas, triazines and organophosphorous species among them. Use of turbulent-flow chromatography columns (TFC, 50 x 1 mm, 30-50 microm particle size) as extraction cartridges enables fast on-line SPE at high sampling flow-rate (5 ml/min). Polymeric and carbon based TFC columns (Oasis HLB, Cyclone, Hypercarb) allow complete extraction with good recoveries from water volumes up to 50 ml. On-line coupling to HPLC is performed with re-mixing of the organic TFC eluate with water in front of the analytical column to ensure efficient band focussing. For fast HPLC analysis, a short monolithic column is applied in combination with highly selective API-MS/MS detection. Matrix effects on the APCI-MS/MS signal were found to be reduced by the system to an acceptable minimum. Limits of detection, determined for 10-ml samples of river water were in the range between 0.4 and 13 ng/l typically, except trifluralin (approximately 280 ng/l), which is less susceptible to ionization under atmospheric pressure conditions. At an enriched water volume of 10 ml, the whole SPE-HPLC-MS/MS procedure requires less than 14 min. The method was successfully applied to the analysis of drinking and surface water samples taken from several sampling sites around the city of Leipzig, Germany. Concentrations measured (maximum: 16 ng/l simazine in river water) were far below the concentration limits scheduled by law.