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Background: Single cell mass spectrometry (scMS) has shown great promise for label free proteomics analysis recently. To present single cell samples for proteomics analysis by MS is not a trivial task. Existing methods rely on robotic liquid handlers to scale up sample preparation throughput. The cost associated with specialized equipment hinders the broad adoption of these workflows, and the sequential sample processing nature limits the ultimate throughput., Results: In this work, we report a parallel sample processing workflow that can simultaneously process 10 single cells without the need of robotic liquid handlers for scMS. This method utilized 3D printed microfluidic devices to form reagent arrays on a glass slide, and a magnetic beads-based streamlined sample processing workflow to present peptides for LC-MS detection. We optimized key operational parameters of the method and demonstrated the quantification consistency among 10 parallel processed samples. Finally, the utility of the method in differentiating cell lines and studying the proteome change induced by drug treatment were demonstrated., Significance: The present method allows parallel sample processing for single cells without the need of expensive liquid handlers, which has great potential to further improve throughput and decrease the barrier for single cell proteomics., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Peng Li reports financial support was provided by the National Institutes of Health and the National Science Foundation., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Capillary vibrating sharp-edge spray ionization (cVSSI) combined with hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has been utilized to characterize different solution-phase DNA conformers including DNA G-quadruplex topologies as well as triplex DNA and duplex DNA. In general, G-quadruplex DNA shows a wide range of protection of hydrogens extending from ~12% to ~21% deuterium incorporation. Additionally, the DNA sequences selected to represent parallel, antiparallel, and hybrid G-quadruplex topologies exhibit slight differences in deuterium uptake levels which appear to loosely relate to overall conformer stability. Notably, the exchange level for one of the hybrid sequence sub topologies of G-quadruplex DNA (24 TTG) is significantly different (compared with the others studied here) despite the DNA sequences being highly comparable. For the quadruplex-forming sequences, correlation analysis suggests protection of base hydrogens involved in tetrad hydrogen bonding. For duplex DNA ~19% deuterium incorporation is observed while only ~16% is observed for triplex DNA. This increased protection of hydrogens may be due to the added backbone scaffolding and Hoogsteen base pairing of the latter species. These experiments lay the groundwork for future studies aimed at determining the structural source of this protection as well as the applicability of the approach for ascertaining different oligonucleotide folds, co-existing conformations, and/or overall conformer flexibility., Competing Interests: Conflicts Disclosure The authors declare the following competing financial S.J.V. and P.L. have co-founded a start-up company, Invibragen Inc., to commercialize technologies involving vibrating sharp-edge spray ionization (VSSI). Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Combining microfluidics with mass spectrometry (MS) analysis has great potential for enabling new analytical applications and simplifying existing MS workflows. The rapid development of 3D printing technology has enabled direct fabrication of microfluidic channels using consumer grade 3D printers, which holds great promise to facilitate the adoption of microfluidic devices by the MS community. However, photo polymerization-based 3D printed devices have an issue with chemical leeching, which can introduce contaminant molecules that may present as isobaric ions and/or severely suppress the ionization of target analytes when combined with MS analysis. Although extra cure and washing steps have alleviated the leeching issue, many such contaminant peaks can still show up in mass spectra. In this work, we report a simple surface modification strategy to isolate the chemical leachates from the channel solution thereby eliminating the contaminant peaks for MS analysis. The channel was prepared by fabricating a layer of polydimethylsiloxane graft followed by wetting the graft using silicone oil. The resulting liquid infused surface (LIS) showed significant reduction in contaminant peaks and improvement in the signal intensity of target analytes. The coating showed good stability after long-term usage (7 days) and long-term storage (∼6 months). Finally, the utility of the coating strategy was demonstrated by printing herringbone microfluidic mixers for studying fast reaction kinetics, which obtained comparable reaction rates to literature values. The effectiveness, simplicity, and stability of the present method will promote the adoption of 3D printed microdevices by the MS community., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Peng Li reports financial support was provided by National Institutes of Health. Kathrine Curtin reports financial support was provided by National Science Foundation., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Huntington's Disease is a fatal neurodegenerative disorder caused by expansion of a glutamine repeat region (polyQ) beyond a critical threshold within exon1 of the huntingtin protein (htt). As a consequence of polyQ expansion, htt associates into a variety of aggregate species that are thought to underlie cellular toxicity. Within cells, htt associates with numerous membranous organelles and surfaces that exert influence on the aggregation process. In particular, the first 17 amino acids at the N-terminus of htt (Nt17) serve as a lipid-binding domain that is intrinsically disordered in bulk solution but adopts an amphipathic α-helical structure upon binding membranes. Beyond this, Nt17 is implicated in initiating htt fibrillization. As the interaction between Nt17 and lipid membranes is likely influenced by lipid properties, the impact of lipid headgroups on htt-exon1 aggregation, membrane activity, and the ability to form protein:lipid complexes was determined. Htt-exon1 with a disease-length polyQ domain (46Q) was exposed to lipid vesicles comprised of lipids with either zwitterionic (POPC and POPE) or anionic (POPG and POPS) headgroups. With zwitterionic head groups, large lipid to peptide ratios were required to have a statistically significant impact on htt aggregation. Anionic lipids enhanced htt fibrillization, even at low lipid:protein ratios, and this was accompanied by changes in aggregate morphology. Despite the larger impact of anionic lipids, htt-exon1(46Q) was more membrane active with zwitterionic lipid systems. The ability of Nt17 to form complexes with lipids was also mediated by lipid headgroups as zwitterionic ionic lipids more readily associated with multimeric forms of Nt17 in comparison with anionic lipids. Collectively, these results highlight the complexity of htt/membrane interactions and the resulting impact on the aggregation process., (Copyright © 2020. Published by Elsevier B.V.)

Ion mobility spectrometry-mass spectrometry (IMS-MS) provides information about the structures of gas-phase ions in the form of a collision cross section (CCS) with a neutral buffer gas. Indicating relative ion size, a CCS value alone is of limited utility. Although such information can be used to propose different conformer types, finer details of structure are not captured. The increased accessibility of IMS-MS measurements with commercial instrumentation in recent years has ballooned its usage in combination with separate measurements to provide enhanced data from which greater structural inferences can be drawn. This short review presents recent outstanding developments in scientific research that employs complementary measurements that when combined with IMS-MS data are used to characterize the structures of a wide range of compounds., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Mounting evidence suggests that pulmonary exposure to nanoparticles (NPs) has a toxic effect on biological systems. A number of studies have shown that exposure to NPs result in systemic inflammatory response, oxidative stress, and leukocyte adhesion. However, significant knowledge gaps exist for understanding the key molecular mechanisms responsible for altered microvasculature function. Utilizing comprehensive LC-MS/MS and comparative proteomic analysis strategies, important proteins related to TiO2 NP exposure in rat plasma have been identified. Molecular pathway analysis of these proteins revealed 13 canonical pathways as being significant (p ≤ 0.05), but none were found to be significantly up or down-regulated (z>|2|). This work lays the foundation for future research that will monitor relative changes in protein abundance in plasma and tissue as a function of post-exposure time and TiO2 NP dosage to further elucidate mechanisms of pathway activation as well as to decipher other affected pathways., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Comparative analyses utilizing collision induced dissociation (CID) and vacuum ultraviolet photodissociation (VUVPD) for seven isobaric disaccharides have been performed in order to differentiate the linkage type and anomeric configuration of the isomers. Although an individual CID spectrum of a disaccharide ion provides information related to its structure, CID does not sufficiently differentiate mixture components due to the identical mass-to-charge values of most of the intense fragments. In contrast to the ambiguity of the CID analyses for the disaccharide mixture, VUVPD (157 nm) generates unique fragments for each disaccharide ion that are useful for distinguishing individual components from the mixture. When combined with a gas-phase ion mobility separation of the ions, the identification of each component from the mixture can be obtained.

A new method is presented for constructing ion mobility distributions of precursor ions based upon the extraction of drift time distributions that are monitored for selected fragment ions. The approach is demonstrated with a recently designed instrument that combines ion mobility spectrometry (IMS) with ion trap mass spectrometry (MS) and ion fragmentation, as shown in a recent publication [J. Am. Soc. Mass Spectrom. 22 (2011) 1477-1485]. Here, we illustrate the method by examining selected charge states of electrosprayed ubiquitin ions, an extract from diesel fuel, and a mixture of phosphorylated peptide isomers. For ubiquitin ions, extraction of all drift times over small mass-to-charge (m/z) ranges corresponding to unique fragments of a given charge state allows the determination of precursor ion mobility distributions. A second example of the utility of the approach includes the distinguishing of precursor ion mobility distributions for isobaric, basic components from commercially available diesel fuel. Extraction of data for a single fragment ion is sufficient to distinguish the precursor ion mobility distribution of cycloalkyl-pyridine derivatives from pyrindan derivatives. Finally, the method is applied for the analysis of phosphopeptide isomers (LFpTGHPESLER and LFTGHPEpSLER) in a mixture. The approach alleviates several analytical challenges that include separation and characterization of species having similar (or identical) m/z values within complex mixtures.

A high-throughput approach for biomolecule analysis is demonstrated for a mixture of peptides from tryptic digest of four proteins as well as a tryptic digests of human plasma. In this method a chip based electrospray autosampler coupled to a hybrid ion mobility (IMS) mass spectrometer (MS) is utilized to achieve rapid sample analysis. This high-throughput measurement is realized by exploiting the direct infusion capability of the chip based electrospray with its rapid sample manipulating capability as well as a high sensitive IMS-MS with a recently developed IMS-IMS separation technique that can be multiplexed to provide greater throughput. From replicate IMS-MS runs of known mixtures, the average uncertainty of peak intensities is determined to be +/-7% (relative standard deviation), and a detection limit in the low attomole range is established. The method is illustrated by analyzing 124 human plasma protein samples in duplicate, a measurement that required 16.5 h. Current limitations as well as implications of the high-throughput approach for complex biological sample analysis are discussed.

Although theoretical systems analysis has been available for over half a century, the recent advent of omic high-throughput analytical platforms along with the integration of individual tools and technologies has given rise to the field of modern systems biology. Coupled with information technology, bioinformatics, knowledge management and powerful mathematical models, systems biology has opened up new vistas in our understanding of complex biological systems. Currently there are two distinct approaches that include the inductively driven computational systems biology (bottom-up approach) and the deductive data-driven top-down analysis. Such approaches offer enormous potential in the elucidation of disease as well as defining key pathways and networks involved in optimal human health and nutrition. The tools and technologies now available in systems biology analyses offer exciting opportunities to develop the emerging areas of personalized medicine and individual nutritional profiling.

The development of a multidimensional approach involving high-performance liquid chromatography (LC), ion mobility spectrometry (IMS) and tandem mass spectrometry is described for the analysis of complex peptide mixtures. In this approach, peptides are separated based on differences in their LC retention times and mobilities (as ions drift through He) prior to being introduced into a quadrupole/octopole/time-of-flight mass spectrometer. The initial LC separation and IMS dispersion of ions is used to label ions for subsequent fragmentation studies that are carried out for mixtures of ions. The approach is demonstrated by examining a mixture of peptides generated from tryptic digestion of 18 commercially available proteins. Current limitations of this initial study and potential advantages of the experimental approach are discussed.

We have studied 150 women undergoing elective Caesarean section under spinal anaesthesia. They were allocated randomly to have a 22-gauge Whitacre, a 25-gauge Whitacre or a 26-gauge Quincke needle inserted into the lumbar subarachnoid space. The groups were compared for ease of insertion, number of attempted needle insertions before identification of cerebrospinal fluid, quality of subsequent analgesia and incidence of postoperative complications. There were differences between groups, but they did not reach statistical significance. Postdural puncture headache (PDPH) was experienced by one mother in the 22-gauge Whitacre group, none in the 25-gauge Whitacre group and five in the 26-gauge Quincke group. Five of the six PDPH occurred after a single successful needle insertion. Seven of the 15 mothers in whom more than two needle insertions were made experienced backache, compared with 12 of the 129 receiving two or less (P < 0.001). We conclude that the use of 22- and 25-gauge Whitacre needles in elective Caesarean section patients is associated with a low incidence of PDPH and that postoperative backache is more likely when more than two attempts are made to insert a spinal needle.

Fifty women in labour were allocated randomly to receive either air or saline to assist in the identification of the extradural space by the loss of resistance technique. A study volume of 4 ml of air or saline was used before 0.5% bupivacaine 8 ml and the spread of analgesia was followed for 30 min. The first segment blocked, time of onset, number of blocked segments and height of block were comparable in the two groups. At 30 min, there were eight patients with an unblocked segment in the air group, compared with two in the saline group (P less than 0.01). All unblocked segments were blocked subsequently by further doses of bupivacaine. We conclude that air is more likely than saline to produce unblocked segments in the initiation of extradural analgesia in labour.

We studied patients scheduled for coronary artery bypass surgery following premedication with lorazepam, morphine and droperidol, using pulse oximetry and serial electrocardiographic (ECG) recordings. Arterial oxygen saturation (SaO2) values were compared with those obtained during two control periods when the patients were awake and asleep. All patients demonstrated progressive arterial oxygen desaturation during the premedication period, statistically significant from both controls (P less than 0.001). Twelve of the 15 patients developed hypoxaemia or severe hypoxaemia which was corrected immediately by administration of oxygen. New ECG changes developed during the premedication period in 33% of patients. It is concluded that additional oxygen should be administered to patients receiving this and similar premedication regimens.