A unique mass spectrometry (MS) method has been developed to determine the negatively charged species in live single cells using the positive ionization mode. The method utilizes dicationic ion-pairing compounds through the miniaturized multifunctional device, the single-probe, for reactive MS analysis of live single cells under ambient conditions. In this study, two dicationic reagents, 1,5-pentanediyl-bis(1-butylpyrrolidinium) difluoride (C5(bpyr)2F2) and 1, 3-propanediyl-bis-(tripropylphosphonium) difluoride (C3(triprp)2F2), were added in the solvent and introduced into single cells to extract cellular contents for real-time MS analysis. The negatively charged (1− charged) cell metabolites, which form stable ion-pairs (1+ charged) with dicationic compounds (2+ charged), were detected in positive ionization mode with a greatly improved sensitivity. We have tentatively assigned 192 and 70 negatively charged common metabolites as adducts with (C5(bpyr)2F2) and (C3(triprp)2F2), respectively, in three separate SCMS experiments in the positive ion mode. The total number of tentatively assigned metabolites is 285 for C5(bpyr)2F2 and 143 for C3(triprp)2F2. In addition, the selectivity of dicationic compounds in the complex formation allows for the discrimination of overlapped ion peaks with low abundances. Tandem (MS/MS) analyses at the single cell level were conducted for selected adduct ions for molecular identification. The utilization of the dicationic compounds in the single-probe MS technique provides an effective approach to the detection of a broad range of metabolites at the single cell level.

Existing single cell mass spectrometry (SCMS) sampling platforms are largely designed to work only with immobilized cells and not the suspended cells isolated from patient samples. Here, we present a novel method that integrates a commercially available cell manipulation system commonly used for in vitro fertilization with the Single-probe SCMS sampling technology. The combined Single-probe SCMS/cell manipulating platform is capable of rapidly analyzing intracellular species in real time from a suspension leukemia cell line. A broad range of molecular species was detected, and species of interest were verified using tandem MS (MS/MS). Experimental results were analyzed utilizing statistical analyses such as principle component analysis (PCA) and t-tests. The developed SCMS/cell manipulation system is a versatile tool to provide rapid single cell analysis of broad types of patient cell samples.

Analyzing cellular constituents on the single-cell level through mass spectrometry (MS) allows for a wide range of compounds to be studied simultaneously. However, there is a need for quantitative single-cell mass spectrometry (qSCMS) methods to fully characterize drug efficacy from individual cells within cell populations. In this study, qSCMS experiments were carried out using the Single-probe MS technique. The method was successfully used to perform rapid absolute quantifications of the anticancer drug irinotecan in individual mammalian cancer cells under ambient conditions in real time. Traditional liquid chromatography/mass spectrometry (LC/MS) quantifications of irinotecan in cell lysate samples were used to compare the results from Singleprobe qSCMS. This technique showcases heterogeneity of drug efficacy on the single-cell level.

Oxysterol-binding protein (OSBP) is a lipid transport and regulatory protein required for the replication of Enterovirus genus viruses, which includes many significant human pathogens. Short-term exposure (i.e., 1−6 h) to a low dose (i.e., 1 nM) of the natural product compound OSW-1 induces a reduction of cellular OSBP levels by ∼90% in multiple different cell lines with no measurable cytotoxicity, defect in cellular proliferation, or global proteome reduction. Interestingly, the reduction of OSBP levels persists multiple days after the low-dose, transient OSW-1 compound treatment is ended and the intracellular OSW-1 compound levels drop to undetectable levels. The reduction in OSBP levels is inherited in multiple generations of cells that are propagated after the OSW-1 compound treatment is stopped. The enduring multiday, multigenerational reduction of OSBP levels triggered by the OSW-1 compound is not due to proteasome degradation of OSBP or due to a reduction in OSBP mRNA levels. OSW-1 compound treatment induces transient autophagy in cells, but blocking autophagy does not rescue OSBP levels. Although the specific cellular mechanism of long-term OSBP repression is not yet identified, these results clearly show the existence of an OSBP specific cellular regulation process that is triggered upon treatment with an OSBP-binding compound. The stable reduction of OSBP levels upon short-term, transient OSW-1 compound treatment will be a powerful tool to understand OSBP regulation and cellular function. Additionally, the persistent reduction in OSBP levels triggered by the transient OSW-1 compound treatment substantially reduces viral replication in treated cells. Therefore, the long-term, compound-induced reduction of OSBP in cells presents a new route to broad spectrum anti-Enterovirus activity, including as a novel route to antiviral prophylactic treatment through small molecule targeting a human host protein.O xysterol-binding protein (OSBP) and the OSBP-related proteins (ORPs) are a family of lipid and sterol binding proteins conserved in all eukaryotes. 1,2 The 12 OSBP/ORP human proteins share a conserved ∼50 kDa, C-terminal ligand binding domain. 1,2 OSBP and ORP4, the member most closely related to OSBP, share substantial sequence similarity, and both contain N-terminal pleckstrin homology (PH) and FFAT domains. 1,2 Individual OSBP/ORP family members are reported to have many different cellular functions, 1,2 including serving as cellular sensors for lipid membrane composition. 3−6 OSBP is reported to be localized at the membrane contact site between the ER and Golgi, and from this location, OSBP is

In clinical cancer medicine, the current inability to quantify intracellular chemotherapy drug concentrations in individual human cells limits the personalization and overall effectiveness of drug administration. New bioanalytical methods capable of real-time measurement of drug levels in live single cancer cells would allow for more adaptive and personalized administration of chemotherapy drugs, potentially leading to better clinical outcomes with fewer side effects. In this study, we report the development of a new quantitative single cell mass spectrometry (qSCMS) method capable of providing absolute drug amounts and concentrations in single cancer cells. Using this qSCMS system, quantitative analysis of the intracellular drug gemcitabine present in individual bladder cancer cells is reported, including in bladder cancer cells isolated from patients undergoing standard-of-care gemcitabine chemotherapy. The development of single cell pharmacology bioanalytical methods can potentially lead to more effective and safely administered drug medications in patients, especially in the treatment of cancer.