Direct sub-atmospheric pressure ionization mass spectrometry: Evaporation/sublimation-driven ionization is amazing, fundamentally, and practically.

This paper covers direct sub-atmospheric pressure ionization mass spectrometry (MS). The discovery, applications, and mechanistic aspects of novel ionization processes for use in MS that are not based on the high-energy input from voltage, laser, and/or high temperature but on sublimation/evaporation within a region linking a higher to lower pressure and modulated by heat and collisions, are discussed, including how this new reality has guided a series of discoveries, instrument developments, and commercialization. A research focus, inter alia, is on how best to understand, improve, and use these novel ionization processes, which convert volatile and nonvolatile compounds from solids (sublimation) or liquids (evaporation) into gas-phase ions for analysis by MS providing reproducible, accurate, sensitive, and prompt results. Our perception on how these unprecedented versus traditional ionization processes/methods relate to each other, how they can be made to coexist on the same mass spectrometer, and an outlook on new and expanded applications (e.g., clinical, portable, fast, safe, and autonomous) is presented, and is based on ST's Opening lecture presentation at the Nordic Mass spectrometry Conference, Geilo, Norway, January 2023. Focus will be on matrix-assisted ionization (MAI) and solvent-assisted ionization (SAI) MS covering the period from 2010 to 2023; a potential paradigm shift in the making.

PXMP4 promotes gastric cancer cell epithelial-mesenchymal transition via the PI3K/AKT signaling pathway.

BACKGROUND: Peroxisomal membrane protein 4 (PXMP4), a member of the peroxisome membrane protein PXMP2/4 family, participates in the progression of several malignant cancers. Nevertheless, the effect of PXMP4 in the development of gastric cancer (GC) is still unknown. As a result, the focus of this investigation was to elucidate the potential mechanisms of PXMP4 in GC. METHODS AND RESULTS: Firstly, bioinformatics analysis results showed higher expression of PXMP4 in GC tissues. Secondly, clinical analysis of 57 patients with GC revealed correlations between PXMP4 expression and differentiation, depth of invasion, as well as TNM stage. Furthermore, individuals with elevated PXMP4 expression in GC exhibited an unfavorable prognosis. In vitro data showed the involvement of knockdown/overexpression of PXMP4 in the proliferation, invasion, and migration of GC cells, and triggering the epithelial-mesenchymal transition (EMT) of GC cells through the activation of the PI3K/AKT signaling pathway. LY294002, a PI3K/AKT inhibitor, inhibited the expression of PI3K/AKT-related proteins but did not affect the expression of PXMP4. CONCLUSIONS: These findings indicate that PXMP4 potentially functions as an upstream molecule in the PI3K/AKT pathway, governing the EMT process in GC.

Single-cell genomic variation induced by mutational processes in cancer.

How cell-to-cell copy number alterations that underpin genomic instability1 in human cancers drive genomic and phenotypic variation, and consequently the evolution of cancer2, remains understudied. Here, by applying scaled single-cell whole-genome sequencing3 to wild-type, TP53-deficient and TP53-deficient;BRCA1-deficient or TP53-deficient;BRCA2-deficient mammary epithelial cells (13,818 genomes), and to primary triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC) cells (22,057 genomes), we identify three distinct 'foreground' mutational patterns that are defined by cell-to-cell structural variation. Cell- and clone-specific high-level amplifications, parallel haplotype-specific copy number alterations and copy number segment length variation (serrate structural variations) had measurable phenotypic and evolutionary consequences. In TNBC and HGSC, clone-specific high-level amplifications in known oncogenes were highly prevalent in tumours bearing fold-back inversions, relative to tumours with homologous recombination deficiency, and were associated with increased clone-to-clone phenotypic variation. Parallel haplotype-specific alterations were also commonly observed, leading to phylogenetic evolutionary diversity and clone-specific mono-allelic expression. Serrate variants were increased in tumours with fold-back inversions and were highly correlated with increased genomic diversity of cellular populations. Together, our findings show that cell-to-cell structural variation contributes to the origins of phenotypic and evolutionary diversity in TNBC and HGSC, and provide insight into the genomic and mutational states of individual cancer cells.

Accurate determination of CRISPR-mediated gene fitness in transplantable tumours.

Assessing tumour gene fitness in physiologically-relevant model systems is challenging due to biological features of in vivo tumour regeneration, including extreme variations in single cell lineage progeny. Here we develop a reproducible, quantitative approach to pooled genetic perturbation in patient-derived xenografts (PDXs), by encoding single cell output from transplanted CRISPR-transduced cells in combination with a Bayesian hierarchical model. We apply this to 181 PDX transplants from 21 breast cancer patients. We show that uncertainty in fitness estimates depends critically on the number of transplant cell clones and the variability in clone sizes. We use a pathway-directed allelic series to characterize Notch signaling, and quantify TP53 / MDM2 drug-gene conditional fitness in outlier patients. We show that fitness outlier identification can be mirrored by pharmacological perturbation. Overall, we demonstrate that the gene fitness landscape in breast PDXs is dominated by inter-patient differences.

Clonal fitness inferred from time-series modelling of single-cell cancer genomes.

Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNAs), has been impeded by lack of time-series single-cell sampling of polyclonal populations and temporal statistical models1-7. Here we generated 42,000 genomes from multi-year time-series single-cell whole-genome sequencing of breast epithelium and primary triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), revealing the nature of CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model8,9 to infer clonal fitness, we found that TP53 mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in TNBC PDX models with mutated TP53, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDXs resulted in cisplatin-resistant clones emerging from low-fitness phylogenetic lineages in the untreated setting. Conversely, high-fitness clones from treatment-naive controls were eradicated, signalling an inversion of the fitness landscape. Finally, upon release of drug, selection pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours.

An overview of biological applications and fundamentals of new inlet and vacuum ionization technologies.

RATIONALE: The developments of new ionization technologies based on processes previously unknown to mass spectrometry (MS) have gained significant momentum. Herein we address the importance of understanding these unique ionization processes, demonstrate the new capabilities currently unmet by other methods, and outline their considerable analytical potential. METHODS: The inlet and vacuum ionization methods of solvent-assisted ionization (SAI), matrix-assisted ionization (MAI), and laserspray ionization can be used with commercial and dedicated ion sources producing ions from atmospheric or vacuum conditions for analyses of a variety of materials including drugs, lipids, and proteins introduced from well plates, pipet tips and plate surfaces with and without a laser using solid or solvent matrices. Mass spectrometers from various vendors are employed. RESULTS: Results are presented highlighting strengths relative to ionization methods of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization. We demonstrate the utility of multi-ionization platforms encompassing MAI, SAI, and ESI and enabling detection of what otherwise is missed, especially when directly analyzing mixtures. Unmatched robustness is achieved with dedicated vacuum MAI sources with mechanical introduction of the sample to the sub-atmospheric pressure (vacuum MAI). Simplicity and use of a wide array of matrices are attained using a conduit (inlet ionization), preferably heated, with sample introduction from atmospheric pressure. Tissue, whole blood, urine (including mouse, chicken, and human origin), bacteria strains and chemical on-probe reactions are analyzed directly and, especially in the case of vacuum ionization, without concern of carryover or instrument contamination. CONCLUSIONS: Examples are provided highlighting the exceptional analytical capabilities associated with the novel ionization processes in MS that reduce operational complexity while increasing speed and robustness, achieving mass spectra with low background for improved sensitivity, suggesting the potential of this simple ionization technology to drive MS into areas currently underserved, such as clinical and medical applications.

Epiclomal: Probabilistic clustering of sparse single-cell DNA methylation data.

We present Epiclomal, a probabilistic clustering method arising from a hierarchical mixture model to simultaneously cluster sparse single-cell DNA methylation data and impute missing values. Using synthetic and published single-cell CpG datasets, we show that Epiclomal outperforms non-probabilistic methods and can handle the inherent missing data characteristic that dominates single-cell CpG genome sequences. Using newly generated single-cell 5mCpG sequencing data, we show that Epiclomal discovers sub-clonal methylation patterns in aneuploid tumour genomes, thus defining epiclones that can match or transcend copy number-determined clonal lineages and opening up an important form of clonal analysis in cancer. Epiclomal is written in R and Python and is available at https://github.com/shahcompbio/Epiclomal.

MicroRNA-384 Inhibits the Progression of Papillary Thyroid Cancer by Targeting PRKACB.

BACKGROUND: Growing evidence shows that dysregulation of miRNAs plays a significant role in papillary thyroid cancer (PTC) tumorigenesis and development. The abnormal expression of miR-384 has been acknowledged in the proliferation or metastasis of some cancers. However, the function and the underlying mechanism of miR-384 in PTC progression remain largely unknown. METHODS: Real-time PCR was conducted to detect miR-384 expression in 58 cases of PTC and their adjacent noncancerous tissues. MTT, soft agar assay Transwell assay, and wound-healing assay were carried out to explore the biological function of miR-384 in PTC cell lines of BCPAP and K1. Bioinformatics analysis, dual-luciferase reporter assay, western blot, and functional complementation analysis were conducted to explore the target gene of miR-384. Moreover, Spearman's correlation analysis was conducted to reveal the correlation between miR-384 and PRKACB mRNA in PTC. RESULTS: The expression of miR-384 decreased obviously in PTC, especially in the tumors with lymph node metastasis or larger tumor size. The ectopic upregulation of miR-384 significantly suppressed PTC progression, and the inhibition of miR-384 had the opposite effects. Moreover, PRKACB gene was confirmed as the target of miR-384. CONCLUSION: The study suggests that miR-384 serves as a tumor suppressor in PTC progression by directly targeting the 3'-UTR of PRKACB gene.

RGMB-AS1/miR-4428/PBX1 axis drives the progression of cervical cancer.

BACKGROUND: RGMB antisense RNA 1 (RGMB-AS1) is a member of long non-coding RNAs (lncRNAs) and relates to the carcinogenesis of numerous cancers. Nonetheless, its performance and mechanism in cervical cancer (CC) is unclear. METHODS: The expressions of RGMB-AS1, microRNA-4428 (miR-4428), PBX homeobox 1 (PBX1) were analyzed by quantitative real-time PCR (qRT-PCR). Nuclear-cytoplasmic fractionation was used to locate RGMB-AS1. Cell counting kit-8 (CCK-8), EdU, TUNEL, Western blot and transwell assays were performed to assess RGMB-AS1 function in proliferation, apoptosis, and invasion in vitro. Interplays involving miR-4428, RGMB-AS1 and PBX1 were verified applying luciferase reporter, RNA pull-down and RNA immunoprecipitation (RIP). RESULTS: RGMB-AS1 level was high in CC specimens and cells. RGMB-AS1 encouraged proliferation, and invasion, and depressed apoptosis in CC cells. Further, miR-4428 was screened as a targeted for RGMB-AS1, and RGMB-AS1 performed the competitive endogenous RNA (ceRNA) role to release PBX1 from miR-4428. Correlation analysis based on clinical specimens confirmed positive association between RGMB-AS1 and PBX1 and negative association of miR-4428 with RGMB-AS1 and PBX1. Rescue experiments indicated that PBX1 overexpression counteracted RGMB-AS1 silence-caused inhibition on CC development. CONCLUSIONS: RGMB-AS1 regulated miR-4428/PBX1 axis to aggravate CC development, indicating that targeting RGMB-AS1 could be a potent way for developing the novel therapeutic methods for CC patients.

Clonal Decomposition and DNA Replication States Defined by Scaled Single-Cell Genome Sequencing.

Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.

clonealign: statistical integration of independent single-cell RNA and DNA sequencing data from human cancers.

Measuring gene expression of tumor clones at single-cell resolution links functional consequences to somatic alterations. Without scalable methods to simultaneously assay DNA and RNA from the same single cell, parallel single-cell DNA and RNA measurements from independent cell populations must be mapped for genome-transcriptome association. We present clonealign, which assigns gene expression states to cancer clones using single-cell RNA and DNA sequencing independently sampled from a heterogeneous population. We apply clonealign to triple-negative breast cancer patient-derived xenografts and high-grade serous ovarian cancer cell lines and discover clone-specific dysregulated biological pathways not visible using either sequencing method alone.

Spontaneous Charge Separation and Sublimation Processes are Ubiquitous in Nature and in Ionization Processes in Mass Spectrometry.

Ionization processes have been discovered by which small and large as well as volatile and nonvolatile compounds are converted to gas-phase ions when associated with a matrix and exposed to sub-atmospheric pressure. Here, we discuss experiments further defining these simple and unexpected processes. Charge separation is found to be a common process for small molecule chemicals, solids and liquids, passed through an inlet tube from a higher to a lower pressure region, with and without heat applied. This charge separation process produces positively- and negatively-charged particles with widely different efficiencies depending on the compound and its physical state. Circumstantial evidence is presented suggesting that in the new ionization process, charged particles carry analyte into the gas phase, and desolvation of these particles produce the bare ions similar to electrospray ionization, except that solid particles appear likely to be involved. This mechanistic proposition is in agreement with previous theoretical work related to ion emission from ice. Graphical Abstract ᅟ.

An LC/MS Method Providing Improved Sensitivity: Electrospray Ionization Inlet.

Electrospray ionization inlet (ESII) combines positive aspects of electrospray ionization (ESI) and solvent-assisted ionization (SAI). Similar to SAI, the analyte solution is directly introduced into a heated inlet tube linking atmospheric pressure and the initial vacuum stage of the mass spectrometer. However, unlike SAI, in ESII a voltage is applied to the solution through a metal union linking two sections of fused silica tubing through which solution flows into the inlet. Here, we demonstrate liquid chromatography (LC) ESII/MS on two different mass spectrometers using a mixture of drugs, a peptide standard mixture, and protein digests. This LC-ESII/MS approach has little dead volume and thus provides excellent chromatographic resolution at mobile phase flow rates from 1 to 55 µL min-1. Significant improvement in ion abundance and less chemical background ions were observed relative to ESI for all drugs and peptides tested at flow rates from 15 to 55 µL min-1. At a low inlet tube temperature, ESII has an ionization selectivity similar to that of ESI but, at higher inlet temperatures, appears to have the attributes of both ESI and SAI.

A broad-based study on hyphenating new ionization technologies with MS/MS for PTMs and tissue characterization.

Matrix-assisted ionization (MAI) is a newly discovered method for converting compounds from the solid phase to gas-phase ions having charge states similar to electrospray ionization (ESI), but without the need for high-energy sources such as lasers or high voltage. Laserspray ionization (LSI) is a subset of MAI that uses a laser to provide high spatial resolution analyses, but the laser is not directly involved in the ionization process. These methods produce multiply-charged analyte ions that are useful for characterizing compounds directly from surfaces using advanced characterization technologies. Because the multiply-charged ions originate from charged matrix clusters, efficient desolvation of the matrix is a prerequisite. Here, we report on the utility of collision-induced dissociation (CID) and electron transfer dissociation (ETD) coupled to mass spectrometry using several MAI and LSI matrices for peptide and protein characterization employing mass spectrometers from two manufacturers. The information obtained is similar to that using ESI for most analyses and superior to matrix-assisted laser desorption/ionization (MALDI) as is shown for intact proteins and protein digests directly from mouse brain tissue sections. The ionization processes are soft so that posttranslational modification (e.g. phosphorylation) sites are readily determined. Instances where ETD or CID in conjunction with MAI failed are attributed to lack of desolvation of charged matrix:analyte particles.

Decreased 11ß-Hydroxysteroid Dehydrogenase 1 Level and Activity in Murine Pancreatic Islets Caused by Insulin-Like Growth Factor I Overexpression.

We have reported a high expression of IGF-I in pancreatic islet ß-cells of transgenic mice under the metallothionein promoter. cDNA microarray analysis of the islets revealed that the expression of 82 genes was significantly altered compared to wild-type mice. Of these, 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1), which is responsible for the conversion of inert cortisone (11-dehydrocorticosterone, DHC in rodents) to active cortisol (corticosterone) in the liver and adipose tissues, has not been identified previously as an IGF-I target in pancreatic islets. We characterized the changes in its protein level, enzyme activity and glucose-stimulated insulin secretion. In freshly isolated islets, the level of 11ß-HSD1 protein was significantly lower in MT-IGF mice. Using dual-labeled immunofluorescence, 11ß-HSD1 was observed exclusively in glucagon-producing, islet α-cells but at a lower level in transgenic vs. wild-type animals. MT-IGF islets also exhibited reduced enzymatic activities. Dexamethasone (DEX) and DHC inhibited glucose-stimulated insulin secretion from freshly isolated islets of wild-type mice. In the islets of MT-IGF mice, 48-h pre-incubation of DEX caused a significant decrease in insulin release, while the effect of DHC was largely blunted consistent with diminished 11ß-HSD1 activity. In order to establish the function of intracrine glucocorticoids, we overexpressed 11ß-HSD1 cDNA in MIN6 insulinoma cells, which together with DHC caused apoptosis and a significant decrease in proliferation. Both effects were abolished with the treatment of an 11ß-HSD1 inhibitor. Our results demonstrate an inhibitory effect of IGF-I on 11ß-HSD1 expression and activity within the pancreatic islets, which may mediate part of the IGF-I effects on cell proliferation, survival and insulin secretion.

Drug detection and quantification directly from tissue using novel ionization methods for mass spectrometry.

Solvent assisted ionization inlet (SAII) and matrix assisted ionization vacuum (MAIV) were used to quantify rapidly an antipsychotic drug, clozapine, directly from surfaces with minimal sample preparation. This simple surface analysis method based on SAII- and MAIV-mass spectrometry (MS) was developed to allow the detection of endogenous lipids, metabolites, and clozapine directly from sections of mouse brain tissue. A rapid surface assessment was achieved by SAII with the assistance of heat applied to the mass spectrometer inlet. MAIV provided an improved reproducibility without the need of a heated inlet. In addition, isotope dilution and standard addition were used without sample clean-up, and the results correlate well with liquid chromatography tandem MS using sample work-up. Using the simple surface methods, standard solutions containing clozapine and a deuterated internal standard (clozapine-d8) at different concentration ratios were used in the extraction and quantification of clozapine from brain tissue sections of a drug-treated mouse using different tissue thicknesses. The amount of clozapine extracted by these surface methods was independent of tissue thickness.

High-throughput characterization of small and large molecules using only a matrix and the vacuum of a mass spectrometer.

Matrix assisted ionization vacuum (MAIV) rapidly generates gas-phase analyte ions from subliming solid-phase matrix:analyte crystals for analysis by mass spectrometry (MS). Ionization from the solid-phase allows the use of a variety of surfaces for introducing matrix:analyte samples to the vacuum of a mass spectrometer, including common laboratory materials, such as disposable pipet tips, filter paper, tooth picks, and nylon mesh. MAIV is shown here to be capable of analyses as fast as 3 s per sample with achievable sensitivities in the low femtomole range. MAIV-MS coupled with ion mobility spectrometry (IMS)-MS and tandem mass spectrometry (MS/MS) is shown to be especially powerful for analysis and characterization of a wide range of molecules ranging from small molecules such as drugs and metabolites (∼300 Da) to intact proteins (25.6 kDa). Automated sample introduction is demonstrated on two different commercial mass spectrometers using a programmable XYZ stage. A MAIV high-throughput nontargeted MS(E) approach is also demonstrated utilizing IMS for rapid characterization of small molecules and peptides from standard solutions, as well as drug spiked human urine.

Gas-phase ions produced by freezing water or methanol for analysis using mass spectrometry.

Introducing water or methanol containing a low concentration of volatile or nonvolatile analyte into an inlet tube cooled with dry ice linking atmospheric pressure and the first vacuum stage of a mass spectrometer produces gas-phase ions even of small proteins that can be detected by mass spectrometry. Collision-induced dissociation experiments conducted in the first vacuum region of the mass spectrometer suggest analyte ions being protected by a solvent cage. The charges may be produced by processes similar to those proposed for charge separation under freezing conditions in thunderclouds. By this process, the surface of an ice pellet is charged positive and the interior negative so that removal of surface results in charge separation. A reversal of surface charge is expected for a heated droplet surface, and this is observed by heating rather than cooling the inlet tube. These observations are consistent with charged supercooled droplets or ice particles as intermediates in the production of analyte ions under freezing conditions.

Matrix-assisted ionization vacuum for high-resolution Fourier transform ion cyclotron resonance mass spectrometers.

Matrix-assisted ionization vacuum (MAIV) produces charge states similar to electrospray ionization (ESI) from the solid state without requiring high voltage or added heat. MAIV differs from matrix-assisted laser desorption/ionization (MALDI) in that no laser is needed and abundant multiply charged ions are produced from molecules having multiple basic sites such as proteins. Here we introduce simple modifications to the commercial vacuum MALDI and ESI sources of a 9.4 T Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer to perform MAIV from both intermediate and atmospheric pressure. The multiply charged ions are shown for the proteins bovine insulin, ubiquitin, and lysozyme using 3-nitrobenzonitrile as matrix. These are the first examples of MAIV operating at pressures as low as 10(-6) mbar in an FT-ICR mass spectrometer source, and the expected mass resolving power of 100000 to 400000 is achieved. Identical protein charge states are observed with and without laser ablation indicating minimal, if any, role of photochemical ionization for the compounds studied.

Toward high spatial resolution sampling and characterization of biological tissue surfaces using mass spectrometry.

Mass spectrometry has emerged as a powerful tool for the bioanalytical sciences because of its ability to characterize small and large biomolecules in vanishingly small amounts. A recurring motif in mass spectrometry aims to decipher the chemical composition of biological samples at the molecular level, requiring drastic improvements in the ability to interrogate well defined and highly spatially resolved areas of a sample surface. With the growth of novel ionization methods, numerous advances have been made in sampling biological tissue surfaces. Here, current advancements in ambient, inlet, and vacuum ionization methods are discussed with respect to the potential improvements in the goal of achieving high spatial resolution and/or fast surface analysis. Of similar importance is the need for improvements in applicable characterization strategies using high performance fragmentation technologies such as electron transfer dissociation and electron capture dissociation directly from surfaces, and gas-phase separation through ion mobility spectrometry and high resolution mass spectrometry.