High Spatial Resolution Imaging of Mouse Pancreatic Islets Using Nanospray Desorption Electrospray Ionization Mass Spectrometry

Nanospray Desorption Electrospray Ionization mass spectrometry imaging (nano-DESI MSI) enables ambient imaging of biological samples with high sensitivity and minimal sample pretreatment. Recently, we developed an approach for constant-distance mode MSI using shear force microscopy to precisely control the distance between the sample and the nano-DESI probe. Herein, we demonstrate the power of this approach for robust imaging of pancreatic islets with high spatial resolution of ∼11 μm. Pancreatic islets are difficult to characterize using traditional mass spectrometry approaches due to their small size (∼100 μm) and molecular heterogeneity. Nano-DESI MSI was used to examine the spatial localization of several lipid classes including phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), phosphatidylinositol (PI), and phosphatidylserine (PS) along with fatty acids and their metabolites (e.g., prostaglandins) in the individual islets and surrounding tissue. Several lipids were found to be substantially enhanced in the islets indicating these lipids may be involved in insulin secretion. Remarkably different distributions were observed for several pairs of Lyso PC (LPC) and PC species differing only by one double bond, such as LPC 18:1 vs LPC 18:0, PC 32:1 vs PC 32:0, and PC 34:2 vs PC 34:1. These findings indicate that minor variations in the fatty acid chain length and saturation have a pronounced effect on the localization of PC and LPC species in pancreatic islets. Interestingly, oxidized PC species observed experimentally were found to be specifically localized to pancreatic islets. These PCs are potential biomarkers for reactive oxygen species in the islets, which could be harmful to pancreatic beta cells. The experimental approach presented in this study will provide valuable information on the heterogeneity of individual pancreatic islets, which is difficult to assess using bulk characterization techniques

Mass spectrometry profiling of pentosan polysulfate sodium (PPS) (ASMS 2017)

Pentosan polysulfate (PPS) is a semisynthetic heterogenous sulfated polysaccharide derived from xylan, the β-1,4-linked polymer of xylose. PPS sold by the brand name Elmiron in United States is taken orally to alleviate pain associated with interstitial cystitis. PPS is a mixture of hundreds or more discrete molecules built from a range of oligoxylose lengths modified with different combinations of functional group modifications, including sulfation, 4-O-methyl-glucuronidylation, acetylation, and others. The overall goal of our research is to develop an approach using MS together with other methods such as NMR to profile PPS at the molecular level. Profiling PPS according to its molecular composition would be invaluable for understanding biological activity, bioavailability, and pharmacokinetics, as well as for quality control.One Elmiron (100 mg PPS) capsule was extracted with 1 ml of HPLC-grade water, and further dilutions were made with this stock solution. Diluted PPS at a concentration of 0.5mg/ml was treated with an ion exchange resin for few hours, centrifuged and the supernatant collected. To this supernatant butylamine (15mM) and hexafluoroisopropanol (60mM) were added as an ion-pair reagent (final pH ~8.5). The treated sample was fractionated on C18 SPE cartridge using acetonitrile (ACN) starting from concentration of 10% up to 100% ACN. Each fraction was individually analyzed by FTICR and IMS-MS both in positive and negative mode. Agilent drift tube-IMS-QTOF MS and home-built drift tube IMS-MS were used to characterize PPS from different lots and locations of production.The mass spectrum obtained from PPS directly dissolved in water is complex and difficult to interpret due in-source fragmentation of sulfated oligosaccharides and presence of multiple metal ion adducts [M+Na]. We have explored the potential of ion-pair reversed phase chromatography to extract and analyze PPS using C18-SPE followed by MS detection using FTICR and IMS. When each eluate was injected directly in FTICR without any chromatographic separation, most of the PPS eluted in fraction containing 10% and 20% ACN. Analysis of mass spectra revealed presence of multiply charged state species, mostly +2, +3 and +4 for data collected in positive mode. Analysis of deconvulated peaks in positive mode displayed abundant neutral loss of 171.03 across the entire MS1 spectrum. This neutral loss of 171.03 units is most likely coming from the group –OSO₃NH₂(CH₂)₃CH₃ from PPS backbone. IMS-MS is capable of separating molecules that have the same mass-to-charge (m/z) ratio but different sizes, shapes or conformations. Therefore it is appealing for separating PPS with different polymerized sizes and different charge states and for reducing the complexity of mass spectra. Low-molecular-weight heparin, another sulfated oligosaccharide, was used as a standard to develop IMS-MS method. Heparin DP10 which has molecular weight around 3000 Da has shown a 2D IMS-MS spectrum with trend lines for charge +2 and +3 and m/z range from 1000 to 2000. Preliminary data of PPS showed 2D IMS-MS profiles with charge states from +1 to +5 and m/z range from 300 to 2500. These results show that IMS-MS can reduce the complexity of sulfated polysaccharide spectra by additional separation of different charge states and polysaccharide sizes. However the spectra are still complex for peak assignment without any pre-treatment. The uses of ion exchange resin and ion-pairs have shown improved sensitivity and separation in IMS-MS.<p></p

HAM-5-GFP shows localization with components of the MAK-2 pathway.

<p>(A) Co-localization of HAM-5-GFP and MAK-2-mCherry during germling communication (arrows). (B) Co-localization of HAM-5-GFP and MEK-2-mCherry during germling communication (arrows). (C) Co-localization of HAM-5-GFP and NRC-1-mCherry during germling communication. NRC-1-mCherry strains show low fluorescence <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783-Dettmann1" target="_blank">[18]</a>. (D) HAM-5-GFP and SO-mCherry do not co-localize during chemotropic interactions, but instead show opposite localization to CAT tips in communicating germlings (arrows). The images on the left are bright field images, fluorescent images on the right. Scale bar  = 10 µM. (E) Co-immunoprecipitation experiments showing an interaction between HAM-5-GFP (210 kD; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783.s004" target="_blank">Figure S4D</a>) and MEK-2-mCherry (82.9 kD; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783.s004" target="_blank">Figure S4C</a>) and NRC-1-mCherry (128 kD; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783.s004" target="_blank">Figure S4C</a>). Input panels show Western blots of immunoprecipitated protein samples from 5 hr-old germlings probed with either anti-GFP (free GFP  = 27 kD; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783.s004" target="_blank">Figure S4D</a>) or anti-mCherry antibodies. The output panel is a Western blot of proteins immunoprecipitated by anti-GFP antibodies (and thus HAM-5-GFP) and probed with anti-mCherry antibodies (detecting MEK-2-mCherry or NRC-1-mCherry).</p

HAM-5-GFP localization in WT and Δ<i>mak-2</i> germlings.

<p>(A) Schematic overview of HAM-5 protein structure. The predicted WD40 domains are shown in grey and the putative coiled coil domains are shown as red bars. The two disordered regions with low complexity are depicted by shaded white boxes. The MAPK phosphorylation site (aa 506) is marked by a blue star, the other two sites showing decreased abundance in treated cells (aa 1288 and 1604) are marked by green stars, and other 13 identified phosphorylation sites (S14, S414, S792, S818, S833, T838, T969, S1085, S1199, T1201, S1202, T1353, S1608) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783-Xiong1" target="_blank">[40]</a> are marked by black stars. The putative MAPK docking site is marked by a yellow line. (B) Localization of HAM-5-GFP to puncta localized to CAT tips during chemotropic interactions between genetically identical cells. HAM-5-GFP showed dynamic localization to CAT tips of germlings with an oscillation of every four min (arrow). HAM-5-GFP also localized to puncta within germlings and near nuclear compartments devoid of HAM-5-GFP (asterisks). The image left is a bright field image. Scale bar  = 10 µM. (C) HAM-5-GFP localized to the sites of contact during germling fusion (arrow). (D) Western blots of WT, WT (<i>ham-5-gfp</i>) and Δ<i>mak-2</i> (<i>ham-5-gfp</i>) germlings with immunoprecipitated HAM-5-GFP probed with anti-GFP antibodies (right panel shows longer run showing higher mobility of HAM-5-GFP in wild type germlings) specifically detecting HAM-5-GFP (210 kD; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783.s004" target="_blank">Figure S4D</a>). Lower panel shows a Western blot with identical samples probed with anti-phospho antibodies that specifically detect phosphorylated serine or threonine residues followed by a proline. (E) Localization of HAM-5-GFP to puncta in Δ<i>mak-2</i> germlings. Some puncta showed localization to germling tips, but which did not oscillate during growth (white arrows). Scale bar  = 10 µM.</p

HAM-5-GFP and MAK-2-mCherry localize to puncta in Δ<i>ham-7</i> and Δ<i>ham-11</i> fusion-deficient germlings.

<p>(A) Western blot of protein samples from 5 hr-old WT, Δ<i>ham-5</i>, Δ<i>ham-7</i> and Δ<i>ham-11</i> germlings probed with anti-p42/44 antibodies, which recognize phosphorylated MAK-1 and MAK-2 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783-Pandey1" target="_blank">[8]</a>. As previously shown <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783-Maddi1" target="_blank">[43]</a>, MAK-1 phosphorylation is reduced in the Δ<i>ham-7</i> mutant. (B) HAM-5-GFP and MAK-2-mCherry localization in WT germlings undergoing chemotropic interactions. Arrows show localization to the CAT tip and to cytoplasmic puncta. (C) HAM-5-GFP and MAK-2-mCherry co-localization in Δ<i>ham-7</i> (<i>ham-5-gfp; mak-2-mCherry</i>) germlings. Note lack of chemotropic interactions. Arrows show puncta of HAM-5-GFP and MAK-2-mCherry in Δ<i>ham-7</i> (<i>ham-5-gfp; mak-2-mCherry</i>) germlings, which are often co-localized at the germ tube tip. (D) HAM-5-GFP and MAK-2-mCherry co-localization in Δ<i>ham-11</i> (<i>ham-5-gfp; mak-2-mCherry</i>) germlings. Note lack of chemotropic interactions. HAM-5-GFP and MAK-2-mCherry co-localized to both cytoplasmic and tip-localized puncta (arrows). The upper left panels are bright field images, the upper right panels show GFP fluorescence, the lower left panels show mCherry fluorescence. Lower right panels show merged images of GFP and mCherry images. Scale bars  = 10 µM.</p

Fig 1CD 7A S4 Raw data mouse elfenbein et al

Raw data from figure 1C, 1D, 7A, and S4

Summary of phosphoproteomics results conducted on Δ<i>mak-2^Q100G</i> mutant.

<p>(A) Overview of FunCat categories of proteins harboring the identified phosphopeptides. The upper bar shows the categories of all the proteins with identified phosphopeptides (3200 phosphopeptides, 1164 proteins), the middle bar shows functional categories of proteins with phosphopeptides that showed higher abundance after inhibition (33 phosphopeptides, 27 proteins) and the lower bar shows FunCat analysis of the proteins with phosphopeptides that showed lower abundance in <i>mak-2^Q100G</i> germlings after treatment with 1NM-PP1 (96 phosphopeptides, 67 proteins). (B) Pie chart showing the relative percentages and absolute numbers of single, double, triple and quadruple phosphosites per peptide. (C) Pie chart showing the relative percentages and absolute numbers of phosphorylated serine, threonine and tyrosine in all peptides found.</p

Figure 5 raw data elfenbein etal

Raw data for Figure

Model for HAM-5-MAK-2/MEK-2/NRC-1 function during chemotropic interactions.

<p>HAM-5 interacts with MAK-2, MEK-2 and NRC-1 and assembles in puncta throughout the germling, some of which are recruited to the CAT tip during chemotropic interactions via interactions with a plasma membrane associated protein (MDP: membrane docking protein). Association of the HAM-5/MAK-2/MEK-2/NRC-1 complex to the CAT tips is associated with signal reception from the partner germling. During this process, HAM-5 is successively phosphorylated by MAK-2 and other kinases, resulting in the disassociation of the complex and termination of the ability to receive signal. Nuclear MAK-2 signaling to the transcription factor PP-1 is not believed to be essential for chemotropic interactions as treatment of germlings with cycloheximide did not disturb oscillation of MAK-2 nor chemotropic behavior <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783-Fleissner2" target="_blank">[19]</a>. The coordinated assembly and disassembly of HAM-5/MAK-2/MEK-2/NRC-1 during communication regulates the tempo of chemotropic interactions between germlings.</p

HAM-5-GFP shows oscillatory localization to fusion points and puncta in hyphae showing chemotropism.

<p>(A) Time course of HAM-5-GFP localization to interacting hyphae prior to cell fusion. HAM-5-GFP localized to the hyphal tip of a homing hyphae (white arrow T = 0; T = 8), followed by a disappearance and localization of HAM-5-GFP at the cell surface in the receptive hypha (white arrow; T = 4). Red arrow shows localization to septa near fusion points. At T = 30, HAM-5 is observed at the site of contact (white arrow). Bright field image is shown in upper left panel; remaining panels show GFP fluorescence. Scale bar  = 50 µM. (B) Graphical representation of relative fluorescence intensity (R.F.I.) of HAM-5-GFP localization to the receptive hypha and the homing hypha over the time course (panel A). × axis shows time (min). (C) Graphical representation of HAM-5-GFP fluorescence of interacting fusion hyphae shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783.s006" target="_blank">Figure S6B</a> over an extended time course. Note that following the fusion event (T = 22 min), HAM-5-GFP puncta co-oscillate in both hyphae for an additional 30 minutes, see (D). <i>y</i> axis shows maximal fluorescence intensity (M.F.I.) while the × axis shows time (min). The time points at which the individual pictures taken at T = 4, T = 8, T = 46 and T = 59 minutes are pointed out in the graph (black arrows) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783.s006" target="_blank">Figure S6</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004783#pgen.1004783.s012" target="_blank">Movie S5</a>). The time of fusion at T = 22 min is marked with a black line. (D) Example of HAM-5-GFP appearing in puncta in both hyphae after fusion at T = 46.</p