Activity-based protein profiling reveals active serine proteases that drive malignancy of human ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) is an understudied poor prognosis subtype of ovarian cancer lacking in effective targeted therapies. Efforts to define molecular drivers of OCCC malignancy may lead to new therapeutic targets and approaches. Among potential targets are secreted proteases, enzymes which in many cancers serve as key drivers of malignant progression. Here, we found that inhibitors of trypsin-like serine proteases suppressed malignant phenotypes of OCCC cell lines. To identify the proteases responsible for malignancy in OCCC, we employed activity-based protein profiling to directly analyze enzyme activity. We developed an activity-based probe featuring an arginine diphenylphosphonate warhead to detect active serine proteases of trypsin-like specificity and a biotin handle to facilitate affinity purification of labeled proteases. Using this probe, we identified active trypsin-like serine proteases within the complex proteomes secreted by OCCC cell lines, including two proteases in common, tissue plasminogen activator and urokinase-type plasminogen activator. Further interrogation of these proteases showed that both were involved in cancer cell invasion and proliferation of OCCC cells and were also detected in in vivo models of OCCC. We conclude the detection of tissue plasminogen activator and urokinase-type plasminogen activator as catalytically active proteases and significant drivers of the malignant phenotype may point to these enzymes as targets for new therapeutic strategies in OCCC. Our activity-based probe and profiling methodology will also serve as a valuable tool for detection of active trypsin-like serine proteases in models of other cancers and other diseases.

Dataset for dose and time-dependent transcriptional response to ionizing radiation exposure.

Exposure to ionizing radiation associated with highly energetic and charged heavy particles is an inherent risk astronauts face in long duration space missions. We have previously considered the transcriptional effects that three levels of radiation (0.3 Gy, 1.5 Gy, and 3.0 Gy) have at an immediate time point (1 hr) post-exposure [1]. Our analysis of these results suggest effects on transcript levels that could be modulated at lower radiation doses [2]. In addition, a time dependent effect is likely to be present. Therefore, in order to develop a lab-on-a-chip approach for detection of radiation exposure in terms of both radiation level and time since exposure, we developed a time- and dose-course study to determine appropriate sensitive and specific transcript biomarkers that are detectable in blood samples. The data described herein was developed from a study measuring exposure to 0.15 Gy, 0.30 Gy, and 1.5 Gy of radiation at 1 hr, 2 hr, and 6 hr post-exposure using Affymetrix® GeneChip® PrimeView™ microarrays. This report includes raw gene expression data files from the resulting microarray experiments representing typical radiation exposure levels an astronaut may experience as part of a long duration space mission. The data described here is available in NCBI's Gene Expression Omnibus (GEO), accession GSE63952.

Transcriptional profile of immediate response to ionizing radiation exposure.

Astronauts participating in long duration space missions are likely to be exposed to ionizing radiation associated with highly energetic and charged heavy particles. Previously proposed gene biomarkers for radiation exposure include phosphorylated H2A Histone Family, Member X (γH2AX), Tumor Protein 53 (TP53), and Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A). However, transcripts of these genes may not be the most suitable biomarkers for radiation exposure due to a lack of sensitivity or specificity. As part of a larger effort to develop lab-on-a-chip methods for detecting radiation exposure events using blood samples, we designed a dose-course microarray study in order to determine coding and non-coding RNA transcripts undergoing differential expression immediately following radiation exposure. The main goal was to elicit a small set of sensitive and specific radiation exposure biomarkers at low, medium, and high levels of ionizing radiation exposure. Four separate levels of radiation were considered: 0 Gray (Gy) control; 0.3 Gy; 1.5 Gy; and 3.0 Gy with four replicates at each radiation level. This report includes raw gene expression data files from the resulting microarray experiments from all three radiation levels ranging from a lower, typical exposure than an astronaut might see (0.3 Gy) to high, potentially lethal, levels of radiation (3.0 Gy). The data described here is available in NCBI's Gene Expression Omnibus (GEO), accession GSE64375.

Thermally-Induced Substrate Release Via Intramolecular Cyclizations of Amino Esters and Amino Carbonates.

The relative cleavage of an alcohol from a panel of amino esters and amino carbonates via intramolecular cyclization was examined as a mechanism for substrate release. Thermal stability at 37 °C was observed only for the 7-membered ring progenitors. Applicability of the approach was illustrated by δ-lactam formation within a poly(dimethylsiloxane) microchannel for release of a captured fluorescent probe.

Kv1.3 channel-blocking immunomodulatory peptides from parasitic worms: implications for autoimmune diseases.

The voltage-gated potassium (Kv) 1.3 channel is widely regarded as a therapeutic target for immunomodulation in autoimmune diseases. ShK-186, a selective inhibitor of Kv1.3 channels, ameliorates autoimmune diseases in rodent models, and human phase 1 trials of this agent in healthy volunteers have been completed. In this study, we identified and characterized a large family of Stichodactyla helianthus toxin (ShK)-related peptides in parasitic worms. Based on phylogenetic analysis, 2 worm peptides were selected for study: AcK1, a 51-residue peptide expressed in the anterior secretory glands of the dog-infecting hookworm Ancylostoma caninum and the human-infecting hookworm Ancylostoma ceylanicum, and BmK1, the C-terminal domain of a metalloprotease from the filarial worm Brugia malayi. These peptides in solution adopt helical structures closely resembling that of ShK. At doses in the nanomolar-micromolar range, they block native Kv1.3 in human T cells and cloned Kv1.3 stably expressed in L929 mouse fibroblasts. They preferentially suppress the proliferation of rat CCR7(-) effector memory T cells without affecting naive and central memory subsets and inhibit the delayed-type hypersensitivity (DTH) response caused by skin-homing effector memory T cells in rats. Further, they suppress IFNγ production by human T lymphocytes. ShK-related peptides in parasitic worms may contribute to the potential beneficial effects of probiotic parasitic worm therapy in human autoimmune diseases.

A potent and Kv1.3-selective analogue of the scorpion toxin HsTX1 as a potential therapeutic for autoimmune diseases.

HsTX1 toxin, from the scorpion Heterometrus spinnifer, is a 34-residue, C-terminally amidated peptide cross-linked by four disulfide bridges. Here we describe new HsTX1 analogues with an Ala, Phe, Val or Abu substitution at position 14. Complexes of HsTX1 with the voltage-gated potassium channels Kv1.3 and Kv1.1 were created using docking and molecular dynamics simulations, then umbrella sampling simulations were performed to construct the potential of mean force (PMF) of the ligand and calculate the corresponding binding free energy for the most stable configuration. The PMF method predicted that the R14A mutation in HsTX1 would yield a > 2 kcal/mol gain for the Kv1.3/Kv1.1 selectivity free energy relative to the wild-type peptide. Functional assays confirmed the predicted selectivity gain for HsTX1[R14A] and HsTX1[R14Abu], with an affinity for Kv1.3 in the low picomolar range and a selectivity of more than 2,000-fold for Kv1.3 over Kv1.1. This remarkable potency and selectivity for Kv1.3, which is significantly up-regulated in activated effector memory cells in humans, suggest that these analogues represent valuable leads in the development of therapeutics for autoimmune diseases.

Endothelial cell culture model for replication of physiological profiles of pressure, flow, stretch, and shear stress in vitro.

The phenotype and function of vascular cells in vivo are influenced by complex mechanical signals generated by pulsatile hemodynamic loading. Physiologically relevant in vitro studies of vascular cells therefore require realistic environments where in vivo mechanical loading conditions can be accurately reproduced. To accomplish a realistic in vivo-like loading environment, we designed and fabricated an Endothelial Cell Culture Model (ECCM) to generate physiological pressure, stretch, and shear stress profiles associated with normal and pathological cardiac flow states. Cells within this system were cultured on a stretchable, thin (∼500 µm) planar membrane within a rectangular flow channel and subject to constant fluid flow. Under pressure, the thin planar membrane assumed a concave shape, representing a segment of the blood vessel wall. Pulsatility was introduced using a programmable pneumatically controlled collapsible chamber. Human aortic endothelial cells (HAECs) were cultured within this system under normal conditions and compared to HAECs cultured under static and "flow only" (13 dyn/cm(2)) control conditions using microscopy. Cells cultured within the ECCM were larger than both controls and assumed an ellipsoidal shape. In contrast to static control control cells, ECCM-cultured cells exhibited alignment of cytoskeletal actin filaments and high and continuous expression levels of ß-catenin indicating an in vivo-like phenotype. In conclusion, design, fabrication, testing, and validation of the ECCM for culture of ECs under realistic pressure, flow, strain, and shear loading seen in normal and pathological conditions was accomplished. The ECCM therefore is an enabling technology that allows for study of ECs under physiologically relevant biomechanical loading conditions in vitro.

Exploiting osmosis for blood cell sorting.

Blood is a valuable tissue containing cellular populations rich in information regarding the immediate immune and inflammatory status of the body. Blood leukocytes or white blood cells (WBCs) provide an ideal sample to monitor systemic changes and understand molecular signaling mechanisms in disease processes. Blood samples need to be processed to deplete contaminating erythrocytes or red blood cells (RBCs) and sorted into different WBC sub-populations prior to analysis. This is typically accomplished using immuno-affinity protocols which result in undesirable activation. An alternative is size based sorting which by itself is unsuitable for WBCs sorting due to size overlap between different sub-populations. To overcome this limitation, we investigated the possibility of using controlled osmotic exposure to deplete and/or create a differential size increase between WBC populations. Using a new microfluidic cell docking platform, the response of RBCs and WBCs to deionized (DI) water was evaluated. Time lapse microscopy confirms depletion of RBCs within 15 s and creation of > 3 µm size difference between lymphocytes, monocytes and granulocytes. A flow through microfluidic device was also used to expose different WBCs to DI water for 30, 60 and 90 s to quantify cell loss and activation. Results confirm preservation of ~100% of monocytes, granulocytes and loss of ~30% of lymphocytes (mostly CD3+/CD4+) with minimal activation. These results indicate feasibility of this approach for monocyte, granulocyte and lymphocyte (sub-populations) isolation based on size.

Microfluidic endothelial cell culture model to replicate disturbed flow conditions seen in atherosclerosis susceptible regions.

Atherosclerotic lesions occur non-randomly at vascular niches in bends and bifurcations where fluid flow can be characterized as "disturbed" (low shear stress with both forward and retrograde flow). Endothelial cells (ECs) at these locations experience significantly lower average shear stress without change in the levels of pressure or strain, which affects the local balance in mechanical stresses. Common in vitro models of atherosclerosis focus primarily on shear stress without accounting for pressure and strain loading. To overcome this limitation, we used our microfluidic endothelial cell culture model (ECCM) to achieve accurate replication of pressure, strain, and shear stress waveforms associated with both normal flow seen in straight sections of arteries and disturbed flow seen in the abdominal aorta in the infrarenal segment at the wall distal to the inferior mesenteric artery (IMA), which is associated with high incidence of atherosclerotic lesion formation. Human aortic endothelial cells (HAECs) were cultured within the ECCM under both normal and disturbed flow and evaluated for cell shape, cytoskeletal alignment, endothelial barrier function, and inflammation using immunofluorescence microscopy and flow cytometry. Results clearly demonstrate quantifiable differences between cells cultured under disturbed flow conditions, which are cuboidal with short and randomly oriented actin microfilaments and show intermittent expression of ß-Catenin and cells cultured under normal flow. However, in the absence of pro-inflammatory stimulation, the levels of expression of activation markers: intra cellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), and vascular endothelial cell growth factor - receptor 2 (VEGF-R2) known to be involved in the initiation of plaque formation were only slightly higher in HAECs cultured under disturbed flow in comparison to cells cultured under normal flow.

Endothelial cell culture model of carotid artery atherosclerosis.

Atherosclerotic lesions form non-randomly at locations in bends and bifurcations where the local flow can be classified as 'disturbed flow' and is associated with low shear stress oscillatory or reciprocating flow. Endothelial cells in vivo are constantly exposed to mechanical stimulation due to hemodynamic loading in the form of pulsatile pressure, cyclic stretch and shear stress to maintain phenotype and control function. In conditions like atherosclerosis, the pressure and strain loading remains the same whereas the local fluid flow behavior and shear stress are altered. Common in vitro models of atherosclerosis focus primarily on shear stress without accounting for pressure and strain loading. To overcome this limitation, we used our microfluidic Endothelial Cell Culture Model (ECCM) to achieve accurate replication of pressure, strain and shear stress waveforms associated with both normal flow seen in straight sections of arteries and disturbed flow seen atherosclerosis lesion susceptible regions. We specifically recreated mechanical stresses associated with the proximal internal carotid which is a major risk factor for stroke. Cells cultured using both conditions show distinct differences in alignment and cytoskeletal organization. In summary we recreated pressure, stretch and shear stress loading seen in straight sections and in the proximal internal carotid in a cell culture compatible platform.

Microfluidic cardiac cell culture model (µCCCM).

Physiological heart development and cardiac function rely on the response of cardiac cells to mechanical stress during hemodynamic loading and unloading. These stresses, especially if sustained, can induce changes in cell structure, contractile function, and gene expression. Current cell culture techniques commonly fail to adequately replicate physical loading observed in the native heart. Therefore, there is a need for physiologically relevant in vitro models that recreate mechanical loading conditions seen in both normal and pathological conditions. To fulfill this need, we have developed a microfluidic cardiac cell culture model (µCCCM) that for the first time allows in vitro hemodynamic stimulation of cardiomyocytes by directly coupling cell structure and function with fluid induced loading. Cells are cultured in a small (1 cm diameter) cell culture chamber on a thin flexible silicone membrane. Integrating the cell culture chamber with a pump, collapsible pulsatile valve and an adjustable resistance element (hemostatic valve) in series allow replication of various loading conditions experienced in the heart. This paper details the design, modeling, fabrication and characterization of fluid flow, pressure and stretch generated at various frequencies to mimic hemodynamic conditions associated with the normal and failing heart. Proof-of-concept studies demonstrate successful culture of an embryonic cardiomyoblast line (H9c2 cells) and establishment of an in vivo like phenotype within this system.

Reevaluation of the phospholipid composition in membranes of adult human lenses by (31)P NMR and MALDI MS.

The phospholipid composition of adult human lens membranes differs dramatically from that of any other mammalian membrane. Due to minimal cell turnover, cells in the nucleus of the human lens may be considered as the longest lived cells in our body. This work reassesses previous assignments of phospholipid (31)P NMR resonances in adult human lenses. The new assignments are based not only on chemical shifts but also on temperature coefficients. By addition of known phospholipids and examination by matrix-assisted laser desorption/ionization mass spectrometry, several misassigned resonances have been corrected. The revised composition reveals the possible presence of ceramide-1-phosphate and dihydroceramide-1-phosphate. Among glycerophospholipids, the most abundant one does not correspond to phosphatidylglycerol but may be due to the lysoform of alkyl-acyl analogs of phosphatidylethanolamine. Besides sphingophospholipids, adult human lens membranes contain significant amounts of ether (1-O-alkyl) glycerophospholipids and their corresponding lysoforms.

Secretome from mesenchymal stem cells induces angiogenesis via Cyr61.

It is well known that bone marrow-derived mesenchymal stem cells (MSCs) are involved in wound healing and regeneration responses. In this study, we globally profiled the proteome of MSCs to investigate critical factor(s) that may promote wound healing. Cysteine-rich protein 61 (Cyr61) was found to be abundantly present in MSCs. The presence of Cyr61 was confirmed by immunofluorescence staining and immunoblot analysis. Moreover, we showed that Cyr61 is present in the culture medium (secretome) of MSCs. The secretome of MSCs stimulates angiogenic response in vitro, and neovascularization in vivo. Depletion of Cyr61 completely abrogates the angiogenic-inducing capability of the MSC secretome. Importantly, addition of recombinant Cyr61 polypeptides restores the angiogenic activity of Cyr61-depleted secretome. Collectively, these data demonstrate that Cyr61 polypeptide in MSC secretome contributes to the angiogenesis-promoting activity, a key event needed for regeneration and repair of injured tissues. J. Cell. Physiol. 219: 563-571, 2009. (c) 2009 Wiley-Liss, Inc.

Balance of S1P1 and S1P2 signaling regulates peripheral microvascular permeability in rat cremaster muscle vasculature.

Sphingosine-1-phosphate (S1P) regulates various molecular and cellular events in cultured endothelial cells, such as cytoskeletal restructuring, cell-extracellular matrix interactions, and intercellular junction interactions. We utilized the venular leakage model of the cremaster muscle vascular bed in Sprague-Dawley rats to investigate the role of S1P signaling in regulation of microvascular permeability. S1P signaling is mediated by the S1P family of G protein-coupled receptors (S1P(1-5) receptors). S1P(1) and S1P(2) receptors, which transduce stimulatory and inhibitory signaling, respectively, are expressed in the endothelium of the cremaster muscle vasculature. S1P administration alone via the carotid artery was unable to protect against histamine-induced venular leakage of the cremaster muscle vascular bed in Sprague-Dawley rats. However, activation of S1P(1)-mediated signaling by SEW2871 and FTY720, two agonists of S1P(1), significantly inhibited histamine-induced microvascular leakage. Treatment with VPC 23019 to antagonize S1P(1)-regulated signaling greatly potentiated histamine-induced venular leakage. After inhibition of S1P(2) signaling by JTE-013, a specific antagonist of S1P(2), S1P was able to protect microvascular permeability in vivo. Moreover, endothelial tight junctions and barrier function were regulated by S1P(1)- and S1P(2)-mediated signaling in a concerted manner in cultured endothelial cells. These data suggest that the balance between S1P(1) and S1P(2) signaling regulates the homeostasis of microvascular permeability in the peripheral circulation and, thus, may affect total peripheral vascular resistance.

Ligand-induced nuclear translocation of S1P(1) receptors mediates Cyr61 and CTGF transcription in endothelial cells.

Sphingosine-1-phosphate (S1P) receptor subtype 1 (S1P(1)), a G-protein coupled receptor (GPCR), regulates many biological activities of endothelial cells (ECs). In this report, we show that S1P(1) receptors are present in the nuclei of ECs by using various biochemical and microscopic techniques such as cellular fractionation, immunogold labeling, and confocal microscopic analysis. Live cell imaging showed that plasma membrane S1P(1) receptors are rapidly internalized and subsequently translocated to nuclear compartment upon S1P stimulation. Utilizing membrane biotinylation technique further supports the notion that nuclear S1P(1) receptors were internalized from plasma membrane S1P(1) after ligand treatment. Moreover, nuclear S1P(1) is able to regulate the transcription of Cyr61 and CTGF, two growth factors functionally important in the regulation of vasculature. Collectively, these data suggest a novel S1P-S1P(1) signaling axis present in the nuclear compartment of endothelial cells, which may regulate biological responses of endothelium.

Up-regulating sphingosine 1-phosphate receptor-2 signaling impairs chemotactic, wound-healing, and morphogenetic responses in senescent endothelial cells.

Vascular endothelial cells (ECs) have a finite lifespan when cultured in vitro and eventually enter an irreversible growth arrest state called "cellular senescence." It has been shown that sphingolipids may be involved in senescence; however, the molecular links involved are poorly understood. In this study, we investigated the signaling and functions of sphingosine 1-phosphate (S1P), a serum-borne bioactive sphingolipid, in ECs of different in vitro ages. We observed that S1P-regulated responses are significantly inhibited and the S1P(1-3) receptor subtypes are markedly increased in senescent ECs. Increased expression of S1P(1) and S1P(2) was also observed in the lesion regions of atherosclerotic endothelium, where senescent ECs have been identified in vivo. S1P-induced Akt and ERK1/2 activation were comparable between ECs of different in vitro ages; however, PTEN (phosphatase and tensin homolog deleted on chromosome 10) activity was significantly elevated and Rac activation was inhibited in senescent ECs. Rac activation and senescent-associated impairments were restored in senescent ECs by the expression of dominant-negative PTEN and by knocking down S1P(2) receptors. Furthermore, the senescent-associated impairments were induced in young ECs by the expression of S1P(2) to a level similar to that of in vitro senescence. These results indicate that the impairment of function in senescent ECs in culture is mediated by an increase in S1P signaling through S1P(2)-mediated activation of the lipid phosphatase PTEN.

Influence of temperature on 31P NMR chemical shifts of phospholipids and their metabolites I. In chloroform-methanol-water.

Spectral overlap of (31)P NMR resonances and the lack of reproducibility in chemical shifts corresponding to phospholipids in organic solvents challenge the accuracy of band assignments and quantification. To alleviate these problems, the use of temperature coefficients is proposed. Changes in temperature enable the resolution of overlapped resonances and provide a facile approach for the computation of temperature coefficients. The coefficients were evaluated for various glycero- and sphingo-phospholipids. Their values suggest that differences in H-bonding between the phosphate and the head groups are responsible for the changes of chemical shift with temperature. Among parent phospholipids, and in addition to sphingomyelin, the smallest temperature coefficient values (closest to zero) were observed for phosphatidylcholine, phosphatidylglycerol, dihydrosphingomyelin, and cardiolipin. The highest values were exhibited by phospholipids with protonated head groups, such as phosphatidylserine and phosphatidylethanolamine. The lowest and, in fact, negative values were measured for phospholipids with an exposed phosphate group: phosphatidic acid, ceramide-1-phosphate, and dihydroceramide-1-phosphate. Diacyl, alkyl-acyl, and alkenyl-acyl phospholipids with the same head group exhibited comparable coefficients but differed slightly in chemical shifts. Compared to their parent glycerophospholipids, all lyso analogs had greater temperature coefficients, possibly due to the presence of an extra OH capable of forming a H-bond with the phosphate group.

PEDF from mouse mesenchymal stem cell secretome attracts fibroblasts.

Conditioned medium (secretome) derived from an enriched stem cell culture stimulates chemotaxis of human fibroblasts. These cells are classified as multipotent murine mesenchymal stromal cells (mMSC) by immunochemical analysis of marker proteins. Proteomic analysis of mMSC secretome identifies nineteen secreted proteins, including extracellular matrix structural proteins, collagen processing enzymes, pigment epithelium-derived factor (PEDF) and cystatin C. Immunodepletion and reconstitution experiments show that PEDF is the predominant fibroblast chemoattractant in the conditioned medium, and immunofluorescence microscopy shows strong staining for PEDF in the cytoplasm, at the cell surface, and in intercellular space between mMSCs. This stimulatory effect of PEDF on fibroblast chemotaxis is in contrast to the PEDF-mediated inhibition of endothelial cell migration, reported previously. These differential functional effects of PEDF toward fibroblasts and endothelial cells may serve to program an ordered temporal sequence of scaffold building followed by angiogenesis during wound healing.

Oxidation-induced changes in human lens epithelial cells. 1. Phospholipids.

Lipid compositional changes in lens epithelial cells (HLE B-3) grown in a hyperoxic atmosphere were studied to determine if oxidation could cause changes in the amount and type of phospholipid similar to those found in vivo with age and cataract. The phosphatidylcholines in HLE B-3 cells were 8 times more unsaturated than the sphingomyelins. Cell viability was the same for cells grown for up to 48 h in a normoxic or hyperoxic atmosphere. Lipid oxidation was about three times higher after growth in a hyperoxic atmosphere compared with cells grown in a normoxic atmosphere. The lack of change in the relative amount of sphingomyelin and the decrease in phosphatidylcholine coupled with the increase in lysophosphatidylcholine support the idea that similar mechanisms may be responsible for the lipid compositional changes in both lens epithelial and fiber cells. It is postulated that lipases eliminate oxidized unsaturated glycerolipids, leaving a membrane increasingly composed of more ordered and more saturated sphingolipids. Oxidative stress leads to changes in membrane composition that are consistent with those seen with age in human epithelial cells. Oxidation-induced epithelial phospholipid change is an area of research that has gone virtually unexplored in the human lens and could be relevant to all cell types and may be important to lens clarity.

In vitro and in situ tracking of choline-phospholipid biogenesis by MALDI TOF-MS.

The quantitative monitoring of newly synthesized species of phosphatidylcholines (PCs) and sphingomyelins (SMs) has been achieved in cultured human lens epithelial cells, both in situ and in vitro, with the use of MALDI TOF-MS. As the cells were cultured with deuterated choline-d(9), new peaks that differed from the hydrogenated species by 9.06 Da appeared in the mass spectra. The initial rates of appearance of all deuterated species of PCs were comparable and 4 times higher than those for SMs. After 12 h, those rates began to decrease for PCs but not for deuterated SMs, whose relative contents continued to increase throughout the 72 h of the experiment. The differences in initial rates are consistent with the reported initial generation of PCs, their subsequent degradation, and transfer of their headgroup, phosphorylcholine, to SMs. To further test the ability of MALDI TOF-MS to quantify changes in phospholipid (PL) metabolic pathways, myriocin, an inhibitor of SM synthesis, was added to the cells. In vitro and in situ results revealed a decrease in SMs and an unexpected increase in some PCs. With the use of other deuterated precursors and in combination with postsource decay or tandem MS/MS, this approach could allow the simultaneous tracking of the biosynthesis of multiple PL classes while providing details on their acyl chains.