Explore the vast range of titles available.

We present a lab-on-a-chip approach for the analysis of secondary metabolites produced in microfluidic droplets by simultaneous epifluorescence microscopy and electrospray ionization mass spectrometry (ESI-MS). The approach includes encapsulation and long-term off-chip incubation of microbes in surfactant-stabilized droplets followed by a transfer of droplets into a microfluidic chip for subsequent analysis. Before the reinjected droplets are spaced and electrosprayed from an integrated emitter into a mass spectrometer, the presence of fluorescent marker molecules is monitored nearly simultaneously with a custom-made portable epifluorescence microscope. This combined fluorescence and MS-detection setup allows the analysis of metabolites and fluorescent labels in a complex biological matrix at a single droplet level. Using hyphae of Streptomyces griseus, encapsulated in microfluidic droplets of ~ 200 picoliter as a model system, we show the detection of in situ produced streptomycin by ESI-MS and the feasibility of detecting fluorophores inside droplets shortly before they are electrosprayed. The presented method expands the analytical toolbox for the discovery of bioactive metabolites such as novel antibiotics, produced by microorganisms.

Accurate identification of prostate cancer in frozen sections at the time of surgery can be challenging, limiting the surgeon’s ability to best determine resection margins during prostatectomy. We performed desorption electrospray ionization mass spectrometry imaging (DESI-MSI) on 54 banked human cancerous and normal prostate tissue specimens to investigate the spatial distribution of a wide variety of small metabolites, carbohydrates, and lipids. In contrast to several previous studies, our method included Krebs cycle intermediates (m/z <200), which we found to be highly informative in distinguishing cancer from benign tissue. Malignant prostate cells showed marked metabolic derangements compared with their benign counterparts. Using the “Least absolute shrinkage and selection operator” (Lasso), we analyzed all metabolites from the DESI-MS data and identified parsimonious sets of metabolic profiles for distinguishing between cancer and normal tissue. In an independent set of samples, we could use these models to classify prostate cancer from benign specimens with nearly 90% accuracy per patient. Based on previous work in prostate cancer showing that glucose levels are high while citrate is low, we found that measurement of the glucose/citrate ion signal ratio accurately predicted cancer when this ratio exceeds 1.0 and normal prostate when the ratio is less than 0.5. After brief tissue preparation, the glucose/citrate ratio can be recorded on a tissue sample in 1 min or less, which is in sharp contrast to the 20 min or more required by histopathological examination of frozen tissue specimens.

Electrospray ionization mass spectrometry (ESI-MS) is nowadays one of the cornerstones of biomolecular mass spectrometry and proteomics. Advances in sample preparation and mass analyzers have enabled researchers to extract much more information from biological samples than just the molecular weight. In particular, relevant for structural biology, noncovalent protein–protein and protein–ligand complexes can now also be analyzed by MS. For these types of analyses, assemblies need to be retained in their native quaternary state in the gas phase. This initial small niche of biomolecular mass spectrometry, nowadays often referred to as “native MS,” has come to maturation over the last two decades, with dozens of laboratories using it to study mostly protein assemblies, but also DNA and RNA-protein assemblies, with the goal to define structure–function relationships. In this perspective, we describe the origins of and (re)define the term native MS, portraying in detail what we meant by “native MS,” when the term was coined and also describing what it does (according to us) not entail. Additionally, we describe a few examples highlighting what native MS is, showing its successes to date while illustrating the wide scope this technology has in solving complex biological questions. Graphical Abstract ᅟ

A triangular-shaped flat plastic substrate probe was prepared for direct electrospray ionization mass spectrometry (ESI–MS) for analysis of untreated chemical and biological samples including liquids (Met-Arg-Phe-Ala peptide, reserpine, and dodecyl aldehyde), solids (biological samples, traditional Chinese medicine), and powders (roasted coffee, rhizoma coptidis, lotus plumule, and Schisandra sphenanthera ). Quantitative analysis of reserpine in water yielded a detection limit of 1 ng mL −1 , dynamic response range within 1–500 ng mL −1 , and linearity of signal response ˃0.9925. Compared to the conventional capillary ESI, this plastic probe ESI offers lower cost of analysis (US $0.0056 per probe), higher sensitivity, lower sample consumption, longer signal duration (>6 min), better reproducibility, signal stability, and higher speed of analysis (<10 s per sample, including sample loading). Overall, the results indicate the potential of ESI–MS based on flat plastic probes as a versatile method for simple, sensitive, and stable analysis of untreated biological sample analysis.

Lateral flow immunoassays (LFIAs) are widely used for rapid food safety screening analysis. Thanks to simplified protocols and smartphone readouts, LFIAs are expected to be increasingly used on-site, even by non-experts. As a typical follow-up in EU regulatory settings, suspect samples are sent to laboratories for confirmatory analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, re-analysis by LC-MS/MS is laborious and time-consuming. In this work, an identification LFIA (ID-LFIA) approach followed by quadrupole-orbitrap MS or triple quadrupole MS/MS analysis is presented. As a proof of concept, a dedicated ID-LFIA strip was developed for the mycotoxin deoxynivalenol (DON) following its initial screening by a commercial smartphone LFIA. The ID-LFIA strip can be simply immersed in the same sample extract used for the smartphone LFIA screening, and next, DON is retrieved from the monoclonal antibody with a dissociation solution consisting of methanol/ammonia. The solution thus obtained was analyzed directly in MS in order to rapidly confirm the presence of DON and any cross-reacting species. The protocol developed is capable of coping with severe ion suppression caused by LFIA buffers and nitrocellulose substrate residues. Initial analysis of blank, spiked, and incurred samples showed that the newly developed ID-LFIA-MS method was able to confirm the presence or absence of mycotoxins in the samples previously analyzed by LFIA and also differentiate between DON and DON 3-glucoside yielding the positive screening result. The concept and technique developed are envisaged to complement on-site screening and confirmation of any low molecular weight contaminant in future food control frameworks.

Integrating the governing chemistry with the genomics and phenotypes of microbial colonies has been a “holy grail” in microbiology. This work describes a highly sensitive, broadly applicable, and cost-effective approach that allows metabolic profiling of live microbial colonies directly from a Petri dish without any sample preparation. Nanospray desorption electrospray ionization mass spectrometry (MS), combined with alignment of MS data and molecular networking, enabled monitoring of metabolite production from live microbial colonies from diverse bacterial genera, including Bacillus subtilis, Streptomyces coelicolor, Mycobacterium smegmatis , and Pseudomonas aeruginosa . This work demonstrates that, by using these tools to visualize small molecular changes within bacterial interactions, insights can be gained into bacterial developmental processes as a result of the improved organization of MS/MS data. To validate this experimental platform, metabolic profiling was performed on Pseudomonas sp. SH-C52, which protects sugar beet plants from infections by specific soil-borne fungi [R. Mendes et al. (2011) Science 332:1097–1100]. The antifungal effect of strain SH-C52 was attributed to thanamycin, a predicted lipopeptide encoded by a nonribosomal peptide synthetase gene cluster. Our technology, in combination with our recently developed peptidogenomics strategy, enabled the detection and partial characterization of thanamycin and showed that it is a monochlorinated lipopeptide that belongs to the syringomycin family of antifungal agents. In conclusion, the platform presented here provides a significant advancement in our ability to understand the spatiotemporal dynamics of metabolite production in live microbial colonies and communities.

Constant mode ambient mass spectrometry imaging (MSI) of tissue sections with high lateral resolution of better than 10 μm was performed by combining shear force microscopy with nanospray desorption electrospray ionization (nano-DESI). Shear force microscopy enabled precise control of the distance between the sample and nano-DESI probe during MSI experiments and provided information on sample topography. Proof-of-concept experiments were performed using lung and brain tissue sections representing spongy and dense tissues, respectively. Topography images obtained using shear force microscopy were comparable to the results obtained using contact profilometry over the same region of the tissue section. Variations in tissue height were found to be dependent on the tissue type and were in the range of 0–5 μm for lung tissue and 0–3 μm for brain tissue sections. Ion images of phospholipids obtained in this study are in good agreement with literature data. Normalization of nano-DESI MSI images to the signal of the internal standard added to the extraction solvent allowed us to construct high-resolution ion images free of matrix effects. Graphical Abstract ᅟ

Capillary zone electrophoresis (CZE) is a powerful tool that is progressively being applied for the separation of monoclonal antibody (mAb) charge variants. Mass spectrometry (MS) is the desired detection method concerning identification of mAb variants. In biopharmaceutical applications, there exist optimized and validated electrolyte systems for mAb variant quantification. However, these electrolytes interfere greatly with the electrospray ionization (ESI) process. Here, a heart-cut CZE–CZE–MS setup with an implemented mechanical four-port valve interface was developed that used a generic ε-aminocaproic acid based background electrolyte in the first dimension and acetic acid in the second dimension. Interference-free, highly precise mass data (deviation less than 1 Da) of charge variants of trastuzumab, acting as model mAb system, were achieved. The mass accuracy obtained (low parts per million range) is discussed regarding both measured and calculated masses. Deamidation was detected for the intact model antibody, and related mass differences were significantly confirmed on the deglycosylated level. The CZE–CZE–MS setup is expected to be applicable to a variety of antibodies and electrolyte systems. Thus, it has the potential to become a compelling tool for MS characterization of antibody variants separated in ESI-interfering electrolytes. Graphical Abstract Two-dimensional capillary zone electrophoresis mass spectrometry for the characterization of intact monoclonal antibody (mAb) charge variants. A generic, but highly electrospray-interfering electrolyte system was used as first dimension for mAb charge variant separation and coupled to a volatile electrolyte system as second dimension via a four-port nanoliter valve. In this way, interference-free and precise mass spectrometric data of separated mAb charge variants, including deamidation products, were obtained

Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.

Over the past decade, mass spectrometry (MS)-based “shotgun lipidomics” has emerged as a powerful tool for quantitative and qualitative analysis of the complex lipids in the biological system. The aim of this critical review is to give the interested reader a concise overview of the current state of the technology, focused on lipidomic analysis by mass spectrometry. The pros and cons, and pitfalls associated with each available “shotgun lipidomics” method are discussed; and the new strategies for improving the current methods are described. A list of important papers and reviews that are sufficient rather than comprehensive, covering all the aspects of lipidomics including the workflow, methodology, and fundamentals is also compiled for readers to follow.