RESUMO
Mass spectrometry (MS) allows for automated analysis of complex samples at high resolution without the need for labeling/derivatization. Liquid atmospheric pressure matrix-assisted laser desorption/ionization (LAP-MALDI) enables rapid sample preparation and MS analysis using microtiter-plate formats and high-performing mass spectrometers. We present a step change in high-speed, large-scale MS sample analysis of peptides at 20 samples/s and an enzymatic assay at 40 samples/s, i.e., an order of magnitude faster than current MS platforms. LAP-MALDI requires only low amounts of sample volume (<2 µL), of which only a fraction (<1%) is typically consumed, and allows for multiplexing and high-speed MS/MS analysis, demonstrated at â¼10 samples/s. Its high ion signal stability and similarity to electrospray ionization enables CVs below 10% and the analysis of multiply charged peptide ions at these extreme speeds. LAP-MALDI MS fulfills the speed requirements for large-scale population diagnostics and compound screening with the potential of analyzing >1 million samples per day.
Assuntos
Pressão Atmosférica , Espectrometria de Massas em Tandem , Íons , Lasers , Peptídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Mass spectrometry (MS) has many advantages as a quantitative detection technology for applications within drug discovery. However, current methods of liquid sample introduction to a detector are slow and limit the use of mass spectrometry for kinetic and high-throughput applications. We present the development of an acoustic mist ionization (AMI) interface capable of contactless nanoliter-scale "infusion" of up to three individual samples per second into the mass detector. Installing simple plate handling automation allowed us to reach a throughput of 100â¯000 samples per day on a single mass spectrometer. We applied AMI-MS to identify inhibitors of a human histone deacetylase from AstraZeneca's collection of 2 million small molecules and measured their half-maximal inhibitory concentration. The speed, sensitivity, simplicity, robustness, and consumption of nanoliter volumes of sample suggest that this technology will have a major impact across many areas of basic and applied research.
Assuntos
Acústica , Inibidores de Histona Desacetilases/análise , Espectrometria de Massas/instrumentação , Inibidores de Histona Desacetilases/química , HumanosRESUMO
Tandem mass spectrometry (MS/MS) is an invaluable experimental tool for providing analytical data supporting the identification of small molecules and peptides in mass-spectrometry-based "omics" experiments. Data-dependent MS/MS (DDA) is a real-time MS/MS-acquisition strategy that is responsive to the signals detected in a given sample. However, in analysis of even moderately complex samples with state-of-the-art instrumentation, the speed of MS/MS acquisition is insufficient to offer comprehensive MS/MS coverage of all detected molecules. Data-independent approaches (DIA) offer greater MS/MS coverage, typically at the expense of selectivity or sensitivity. This report describes data-set-dependent MS/MS (DsDA), a novel integration of MS1-data processing and target prioritization to enable comprehensive MS/MS sampling during the initial MS-level experiment. This approach is guided by the premise that in omics experiments, individual injections are typically made as part of a larger set of samples, and feedback between data processing and data acquisition can allow approximately real-time optimization of MS/MS-acquisition parameters and nearly complete MS/MS-sampling coverage. Using a combination of R, Proteowizard, XCMS, and WRENS software, this concept was implemented on a liquid-chromatograph-coupled quadrupole time-of-flight mass spectrometer. The results illustrate comprehensive MS/MS coverage for a set of complex small-molecule samples and demonstrate a strong improvement on traditional DDA.
Assuntos
Análise de Dados , Espectrometria de Massas em Tandem , Animais , Bovinos , Hordeum/química , Músculos/química , Cebolas/químicaRESUMO
Concerted tandem and traveling wave ion mobility mass spectrometry (CTS analysis) is a unique method that results in a four-dimensional data set including nominal precursor ion mass, product ion mobility, accurate mass of product ion, and ion abundance. This nontargeted lipidomics CTS approach was applied in both positive- and negative-ion mode to phospholipids present in human serum, and the data set was used to evaluate the value of product ion mobility in identifying lipids in a complex mixture. It was determined that the combination of diagnostic product ions and unique collisional cross-section values of product ions is a powerful tool in the structural identification of lipids in a complex biological sample.
Assuntos
Fosfolipídeos/sangue , Humanos , Espectrometria de Massas , Estrutura Molecular , Espectrometria de Massas em TandemRESUMO
Mass spectrometry has emerged as a useful tool in the study of proteins and protein complexes. It is of fundamental interest to explore how the structures of proteins and protein complexes are affected by the absence of solvent and how this alters with increasing time in the gas phase. Here we demonstrate that a range of protein and protein complexes can be confined within the Trap T-wave region of a modified Waters Synapt G2S instrument, including monomeric (ß-lactoglobulin), dimeric (ß-lactoglobulin and enolase), tetrameric (streptavidin, concanavalin A, and pyruvate kinase), and pentameric (C-reactive protein) complexes, ranging in size up to 237 kDa. We demonstrate that complexes can be confined within the Trap region for varying lengths of time over the range 1-60 s and with up to 86% trapping efficiency for 1 s trapping. Furthermore, using model systems, we show that these noncovalent complexes can also be fragmented by surface-induced dissociation (SID) following trapping. SID reveals similar dissociation patterns over all trapping times studied for unactivated protein complexes, suggesting that any conformational changes occurring over this time scale are insufficient to cause substantial differences in the SID spectra of these complexes. Intentional alteration of structure by cone activation produces a distinct SID spectrum, with the differences observed being conserved, in comparison to unactivated complex, after trapping. However, subtle differences in the SID spectra of the activated complex are also observed as a function of trapping time.
Assuntos
Proteína C-Reativa/química , Concanavalina A/química , Gases/química , Lactoglobulinas/química , Fosfopiruvato Hidratase/química , Piruvato Quinase/química , Estreptavidina/química , Espectrometria de Massas , Fosfopiruvato Hidratase/metabolismo , Piruvato Quinase/metabolismo , Propriedades de SuperfícieRESUMO
It is critical to the success of any chromatography-based assay that the performance of the LC instrument be checked for its readiness and ability to perform the intended analysis. This includes gaging the suitability of a system to fulfill the purpose of different types of methods. One type of analysis that requires special consideration is the analysis of compounds which are prone to a particular form of non-specific binding, namely metal adsorption, where analytes interact and potentially adsorb to metal contained within the chromatographic flow path. For an analysis of compounds which are susceptible to metal adsorption, ideally a system suitability test would be performed to ensure there will not be any sample loss or detrimental peak shape effects resulting from potential analyte-to-metal interactions. To help chromatographers assess system inertness concerns like this, we have developed a method of testing LC systems for metal interactions using adenosine 5'-(α,ß-methylene)diphosphate (AMPcP). This nucleotide analog has been confirmed to have a propensity to adsorb to titanium and stainless-steel frits and is resistant to hydrolysis and stable to long-term storage and repeat use (as is befitting of any reagent proposed for system suitability testing). AMPcP has been used in a flow injection test (no column in-line) to monitor for losses in recovery and peak shape perturbations that can potentially be present in any chromatography system manufactured with one or more metal based components. In this approach, sequential injections of AMPcP were made without a column and various peak attributes were monitored and ultimately correlated to the amount of metal surface area in the flow path. The ability of this method to discriminate between inert chromatographic surfaces versus exposed metal surfaces was verified by comparing peak areas, peak shapes, and injection repeatability for AMPcP using a UHPLC equipped with MP35N metal alloy components versus an equivalent UHPLC equipped with an ethylene bridged hybrid organic-inorganic surface (or so-called hybrid surface technology). Injections of caffeine were also explored to establish a negative control for this system suitability measurement. Caffeine does not interact with metal surfaces and can therefore give an instrument specific representation of peak shape and dispersion as well as an indication of overall mechanical system performance. Additionally, replicate injections of AMPcP and caffeine onto a UHPLC partially configured with hybrid surface technology (HST) readily identified exposed metal surfaces through an increased peak area relative standard deviation as well as a reduction in absolute recovery. Finally, a novel visualization tool was developed to provide an alternative method of determining system inertness without having to perform chromatographic calculations but instead a graphical peak shape comparison between a negative control, caffeine, and the metal sensitive AMPcP test probe.
Assuntos
Metais , Aço Inoxidável , Adsorção , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Metais/química , Aço Inoxidável/químicaRESUMO
Desorption electrospray ionisation mass spectrometry (DESI-MS) can image hundreds of molecules in a 2D tissue section, making it an ideal tool for mapping tumour heterogeneity. Tumour lipid metabolism has gained increasing attention over the past decade; and here, lipid heterogeneity has been visualised in a glioblastoma xenograft tumour using 3D DESI-MS imaging. The use of an automatic slide loader automates 3D imaging for high sample-throughput. Glioblastomas are highly aggressive primary brain tumours, which display heterogeneous characteristics and are resistant to chemotherapy and radiotherapy. It is therefore important to understand biochemical contributions to their heterogeneity, which may be contributing to treatment resistance. Adjacent sections to those used for DESI-MS imaging were used for H&E staining and immunofluorescence to identify different histological regions, and areas of hypoxia. Comparing DESI-MS imaging with biological staining allowed association of different lipid species with hypoxic and viable tissue within the tumour, and hence mapping of molecularly different tumour regions in 3D space. This work highlights that lipids are playing an important role in the heterogeneity of this xenograft tumour model, and DESI-MS imaging can be used for lipid 3D imaging in an automated fashion to reveal heterogeneity, which is not apparent in H&E stains alone.
Assuntos
Glioblastoma/diagnóstico por imagem , Imageamento Tridimensional/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Diagnóstico por Imagem/métodos , Modelos Animais de Doenças , Feminino , Xenoenxertos/diagnóstico por imagem , Metabolismo dos Lipídeos/fisiologia , Lipídeos/química , Camundongos , Camundongos Nus , Estudo de Prova de Conceito , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Staphylococcus epidermidis is both a human skin commensal and an opportunistic pathogen, causing infections linked to implanted medical devices. This paper describes localized tufts of fibrillar appendages on a subpopulation (25%) of wild-type (WT) S. epidermidis NCTC 11047 cells. The fibrils (122.2 +/- 10.8 nm long) are usually in a lateral position on the cells. Fibrillar (Fib(+)) and nonfibrillar (Fib(-)) subpopulations were separated (enriched) by 34 sequential partitions of WT cells between a buffer phase and a hexadecane phase. Following enrichment, hydrophobic cells from the hexadecane phase comprised 70% Fib(+) cells and the less hydrophobic cells from the buffer phase entirely comprised Fib(-) cells. The Fib(+) and Fib(-) subpopulations did not revert on subculture (34 times) on solid medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell surface proteins from WT, Fib(+), and Fib(-) cells revealed two high-molecular-mass proteins (280 kDa and 230 kDa) on the WT and Fib(+) cells that were absent from the Fib(-) cells. Amino acid sequencing revealed that fragments of both the 280- and 230-kDa proteins had 100% identity to the accumulation-associated protein (Aap). Aap is known to cause biofilm formation if it is truncated by loss of the terminal A domain. Immunogold staining with anti-Aap antibodies labeled tuft fibrils of the WT and Fib(+) cells but not the cell surface of Fib(-) cells. The tufts were labeled with N-terminally directed antibodies (anti-A domain), showing that the fibrillar Aap was not truncated on the cell surface. Thus, the presence of full-length Aap correlated with the low biofilm-forming abilities of both WT and Fib(+) S. epidermidis NCTC 11047 populations. Reverse transcription-PCR showed that aap was transcribed in both Fib(+) and Fib(-) cells. We therefore propose that full-length Aap is expressed on cells of S. epidermidis NCTC 11047 as tufts of short fibrils and that fibril expression is regulated at a posttranscriptional level.