16 research outputs found
Near-Complete Structural Characterization of Phosphatidylcholines Using Electron Impact Excitation of Ions from Organics
Although
lipids are critical components of many cellular assemblies
and biological pathways, accurate descriptions of their molecular
structures remain difficult to obtain. Many benchtop characterization
methods require arduous and time-consuming procedures, and multiple
assays are required whenever a new structural feature is probed. Here,
we describe a new mass-spectrometry-based workflow for enhanced structural
lipidomics that, in a single experiment, can yield almost complete
structural information for a given glycerophospholipid (GPL) species.
This includes the lipid’s sum (Brutto) composition from the
accurate mass measured for the intact lipid ion and the characteristic
headgroup fragment, the regioisomer composition from fragment ions
unique to the sn-1 and sn-2 positions, and the positions of carbon–carbon
double bonds in the lipid acyl chains. Here, lipid ions are fragmented
using electron impact excitation of ions from organics (EIEIO)î—¸a
technique where the singly charged lipid ions are irradiated by an
electron beam, producing diagnostic product ions. We have evaluated
this methodology on various lipid standards, as well as on a biological
extract, to demonstrate this new method’s utility
Distinguishing Cis and Trans Isomers in Intact Complex Lipids Using Electron Impact Excitation of Ions from Organics Mass Spectrometry
We present a mass
spectrometry-based method for the identification
of cis and trans double-bond isomers within intact complex lipid mixtures
using electron impact excitation of ions from organics (EIEIO) mass
spectrometry. EIEIO involves irradiating singly charged lipid ions
with electrons having kinetic energies of 5–16 eV. The resulting
EIEIO spectra can be used to discern cis and trans double-bond isomers
by virtue of the differences in the fragmentation patterns at the
carbon–carbon single bonds neighboring the double bonds. For
trans double bonds, these characteristic fragments include unique
closed-shell and open-shell (radical) products. To explain this fragmentation
pattern in trans double bonds, we have proposed a reaction mechanism
involving excitation of the double bond’s π electrons
followed by hydrogen atom rearrangement. Several lipid standards were
analyzed using the EIEIO method, including mixtures of these standards.
Prior to EIEIO, some of the lipid species in these mixtures were separated
from their isomeric forms by using differential mobility spectrometry
(DMS). For example, mixed cis and trans forms of triacylglycerols
and phosphatidylcholines were identified by this DMS–EIEIO
workflow. With this combined gas-phase separation and subsequent fragmentation,
we could eliminate the need for authentic standards for identification.
When DMS could not separate cis and trans isomers completely, as was
the case with sphingomyelins, we relied upon the aforementioned diagnostic
EIEIO fragment peaks to determine the relative contribution of the
trans double-bond isomer in the mixed samples. We also applied the
DMS–EIEIO methodology to natural samples extracted from a ruminant
(bovine), which serve as common origins of trans fatty acids in a
typical Western diet that includes dairy products
Glycopeptide Identification Using Liquid-Chromatography-Compatible Hot Electron Capture Dissociation in a Radio-Frequency-Quadrupole Ion Trap
We developed a liquid chromatography (LC) compatible
electron capture
dissociation (ECD) mass spectrometer for glycoproteomics, with which
ECD and hot ECD (HECD) experiments can be flexibly switched by quickly
changing the electron energy without further tuning of the mass spectrometer.
Desialylated glycopeptides were dissociated well in both ECD and HECD
experiments. For sialylated glycopeptides, on the other hand, ECD
with electron energy higher than 4 eV showed significantly higher
sequence coverage than that with an electron energy of 0.2 eV. A nano
LC system was coupled to our ECD mass spectrometer to investigate
N-linked glycopeptides from lysylendopeptidase (Lys-C) digests of
human transferrin. ECD spectra at multiple electron energies of 0.2,
5.0, and 9.0 eV were obtained for each targeting precursor ion in
a single LC injection. Glycopeptides with a sialylated bi-, tri-,
or tetra-antennary complex N-glycan were identified with high sequence
coverage by HECD. Glycopeptides with tri- or tetra-antennary N-glycans
have seldom been analyzed by ECD or ETD before this report. We also
found that a preferential dissociation of nonreducing termini of glycans
in glycopeptides by ECD and HECD
Expression of EGFP induced by FLE in fetal and postnatal gonads.
<p>The gonads (ovary and testis) of mFLE-EGFP transgenic mice were prepared in E15.5 (A and B, n = 2), P7 (C and G, n = 4), P14 (D and H, n = 2), P21 (E and I, n = 4), and adult stage at P42 or P56 (F and J, n = 4). Whole views of the gonads are shown. B is a fluorescence view of A. The ovaries are delineated by broken lines. Scale bars = 500 ÎĽm.</p
Overlapped expression of EGFP driven by BAC-Ad4BP and mCherry by mFLE.
<p>Ad4BP-BAC-EGFP transgenic mice were crossed with mFLE-mCherry transgenic mice to generate double transgenic mice (n = 3). Fluorescence views of the adult ovary are shown (A-C). The ovaries were subjected to immunofluorescence with the antibodies for EGFP (green in D and G) and mCherry (red in E and H). Merged views of EGFP and mCherry are shown in F and I, which are further stained with DAPI (blue). Arrows in F and I indicate theca cells (t) or the interstitial gland (ig). Insets are enlarged views of the areas enclosed by rectangles. t, theca cells; ig, interstitial gland. Scale bars for A-C = 500 ÎĽm and those for D-I = 100 ÎĽm.</p
Distribution of EGFP-positive cells in mFLE-EGFP gonads.
<p>mFLE-EGFP transgenic mouse ovaries at P7 (A, n = 4), P14 (B, n = 2), P21 (C, n = 4), and adult stage at P42 or P56 (D, F, G-I, M-O, n = 4), and testes at E18.5 (E) and adult stage at P42 or P56 (J-L, P-R) were sectioned and subjected to immunofluorescence with antibodies for EGFP (green), Ad4BP/SF-1 (green in E, and red in others), and 3β-HSD (red). Merged images for Ad4BP/SF-1 and 3β-HSD (E, F), EGFP and Ad4BP/SF-1 (I, L), and EGFP and 3β-HSD (O, R) are shown. A-F, I, L, O, and R were further stained with DAPI (blue). Arrows in E indicate Sertoli cells (Se) or Leydig cells (Le). Arrows in F, I, L, O, and R indicate theca cells (t) or the interstitial gland (ig). Closed white arrowheads in G-L indicate cells double positive for EGFP and Ad4BP/SF-1, while those in M-R indicate cells double positive for EGFP and 3β-HSD. Open arrowheads in G-L indicate single positive cells for Ad4BP/SF-1, while those in M-R indicate single positive cells for 3β-HSD. Insets are enlarged views of the areas enclosed by rectangles. t, theca cells; ig, interstitial gland; gr, granulosa cells; Le, Leydig cell; Se, Sertoli cell; tc, testicular cord; is, interstitial space. Scale bars = 100 μm.</p
Expression of EGFP induced by Ad4BP-BAC-EGFP.
<p>The ovary (A), testis (B), adrenal gland (C), pituitary (D), and VMH (E), in which endogenous <i>Ad4BP/SF-1</i> is expressed, were prepared from adult Ad4BP-BAC-EGFP transgenic mice (n = 3). EGFP expression was observed under a fluorescent microscope. The testis (F-H), adrenal gland (I-K), pituitary (L-N), VMH (O-Q), and spleen (R-T) were sectioned, followed by immunofluorescence with antibodies for EGFP (green in F, I, L, O, and R) and Ad4BP/SF-1 (red in G, J, M, P, and S). Merged views of EGFP and Ad4BP/SF-1 are shown in H, K, N, Q, and T, which are further stained with DAPI (blue). Insets are enlarged views of the areas enclosed by rectangles. st, seminiferous tubule; adc, adrenal cortex; ess, endothelial cell of splenic sinus. Scale bars in A-E = 500 ÎĽm and those in F-T = 100 ÎĽm.</p
Distribution of EGFP-positive cells in Ad4BP-BAC-EGFP ovary.
<p>The adult ovaries at P42 or P56 were prepared (n = 3) and subjected to immunofluorescence with the antibodies for EGFP (green in A-C and J-L), Ad4BP/SF-1 (red in D-F), and 3β-HSD (red in M-O). Merged views for EGFP and Ad4BP/SF-1 are shown in G, H and I, while those for EGFP and 3β-HSD are shown in P, Q, and R; these are stained simultaneously with DAPI (blue). Arrows in G, H, P and Q indicate theca cells (t) or the interstitial gland (ig). Insets are enlarged views of the areas enclosed by rectangles. f, follicle; t, theca cells; ig, interstitial gland; cl, corpus luteum. Scale bars = 100 μm.</p
Construction of BAC transgene.
<p>Construction of the BAC transgene is shown. The BAC clone (RP23-354G20) used in this study contained <i>Nr6a1</i>, <i>Gpr144-ps</i>, and <i>Psmb7</i> genes in addition to <i>Ad4BP/SF-1</i> as indicated at the top. The directions of gene transcription are indicated by arrows. <i>Ad4BP/SF-1</i> in the BAC was replaced using the targeting vector by recombination using the 5’ and 3’ arms. Ad4BP-BAC-EGFP was finally obtained by FLP-mediated deletion of the 3’ segment of the targeting vector integrated into the BAC. Detailed procedures are described in the Materials and Methods. <i>Nr6a1</i>, nuclear receptor subfamily 6, group A, member 1 (<i>Gcnf</i>); <i>Gpr144-ps</i>, G protein-coupled receptor 144, pseudogene; <i>Psmb7</i>, proteasome subunit, beta type 7; Neo, neomycin resistant gene; pA, poly A; FRT, flippase recombination target; FLP, flippase; EGFP, enhanced green fluorescence protein.</p
Electron Capture Dissociation in a Branched Radio-Frequency Ion Trap
We
have developed a high-throughput electron capture dissociation (ECD)
device coupled to a quadrupole time-of-flight mass spectrometer using
novel branched radio frequency ion trap architecture. With this device,
a low-energy electron beam can be injected orthogonally into the analytical
ion beam with independent control of both the ion and electron beams.
While ions and electrons can interact in a “flow-through”
mode, we observed a large enhancement in ECD efficiency by introducing
a short ion trapping period at the region of ion and electron beam
intersection. This simultaneous trapping mode still provides up to
five ECD spectra per second while operating in an information-dependent
acquisition workflow. Coupled to liquid chromatography (LC), this
LC-ECD workflow provides good sequence coverage for both trypsin and
Lys C digests of bovine serum albumin, providing ECD spectra for doubly
charged precursor ions with very good efficiency