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In JoVE (1)
- Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry
Other Publications (22)
- The Analyst
- Methods in Molecular Biology (Clifton, N.J.)
- Analytical Chemistry
- Journal of Chromatography. A
- Rapid Communications in Mass Spectrometry : RCM
- Analytical Chemistry
- Journal of Chromatography. A
- Journal of Chromatography. A
- Analytica Chimica Acta
- Analytica Chimica Acta
- Analytical Chemistry
- The Analyst
- Journal of Chromatography. A
- The Analyst
- Journal of Agricultural and Food Chemistry
- PloS One
- Rapid Communications in Mass Spectrometry : RCM
- Frontiers in Neuroanatomy
- Journal of Agricultural and Food Chemistry
- International Journal of Proteomics
Articles by Ken K-C Yeung in JoVE
Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry
Sarah Caughlin1, Dae Hee Park1, Ken K-C Yeung2, David F. Cechetto1, Shawn N. Whitehead1
1Department of Anatomy and Cell Biology, The University of Western Ontario, 2Department of Chemistry and Biochemistry, The University of Western Ontario
Other articles by Ken K-C Yeung on PubMed
The Analyst. Jun, 2003 | Pubmed ID: 12866868
The use of surfactants as additives was demonstrated for the first time in capillary isoelectric focusing (CIEF) to dynamically modify the surfaces of bare fused silica capillaries. These surfactants were zwitterionic sulfobetaines: dodecyldimethyl (3-sulfopropyl) ammonium hydroxide (C12N3SO3), hexadecyldimethyl (3-sulfopropyl) ammonium hydroxide (C16N3SO3) and coco (amidopropyl)hydroxyldimethylsulfobetaine (Rewoteric AM CAS U). They were added directly to the protein-ampholyte mixture, and remained in the capillary during isoelectric focusing and mobilization. The C16N3SO3 and CAS U coatings were shown effective in CEF. Separation of seven IEF protein standards was obtained, with significantly improved resolution compared to that from an uncoated silica capillary. The effect of these surfactants on the electroosmotic flow (EOF) in CIEF was determined. CAS U was effective in suppressing the EOF at neutral and alkaline pH conditions, C16N3SO3 was effective in suppressing EOF at acidic and neutral pH conditions. C12N3SO3 however had little effect on the EOF. The pH gradients formed inside these surfactant coated capillaries were recta-linear at pH 6 to 9 (R2 approximately equal to 0.99). Reproducibility of migration time and peak area was determined. For all three coatings, the migration time standard deviations were less than 1.6 min, and the relative standard deviations of area were below 10%. The protein recovery in the CAS U-modified capillary was quantitative or near-quantitative for five of the seven proteins studied.
Methods in Molecular Biology (Clifton, N.J.). 2004 | Pubmed ID: 15163850
Surfactants such as didodecyldimethyl ammonium bromide (DDAB) and 1,2-dilauroyl-sn-phosphatidylcholine (DLPC) form bilayers at the walls of bare silica capillaries. Once formed, these bilayers are stable in the absence of surfactant in the buffer. DDAB provides a cationic bilayer coating which yields a strong reversed EOF and is effective for separation of cationic proteins. DLPC provides a zwitterionic bilayer coating which is effective for both cationic and anionic proteins. The electroosmotic flow (EOF) is strongly suppressed in DLPC-coated capillaries, thus low mobility proteins are slow to elute, and so the coating is favored for separation of high mobility proteins.
Analytical Chemistry. Nov, 2004 | Pubmed ID: 15538809
A simple way to selectively isolate peptides based on their isoelectric points (pI) for MALDI mass spectral analysis is described. An applied voltage was used to electromigrate peptides into a capillary. The capillary was modified with a zwitterionic surfactant, 1,2-dilauroyl-sn-phosphatidylcholine (DLPC), to suppress the electroosmotic flow (EOF) during injection. Hence, either the cationic or the anionic peptides were introduced, depending on the voltage polarity. By controlling the pH, selective loading of peptides was performed to isolate trace components from a mixture. The injected sample plugs were subsequently spotted in nanoliter volumes for MALDI-MS analysis. No significant sample losses resulting from selective sampling were detected. Low attomole-level detection of peptides (adrenocorticotropic hormone fragment 18-39, pI 4.25) was achieved from a mixture containing other peptides (angiotensin I, pI 6.92, and bradykinin, pI 12.00) at 100 000-fold higher concentrations.
Over 1000-fold Protein Preconcentration for Microliter-volume Samples at a PH Junction Using Capillary Electrophoresis
Journal of Chromatography. A. May, 2005 | Pubmed ID: 15909520
An effective protein preconcentration technique specifically designed for microliter-volume samples is presented. The preconcentration is based on the capturing of protein in its isoelectric point (pI) within an applied electric field, using a pH junction created by a discontinuous buffer system. The buffers were chosen to selectively preconcentrate proteins of neutral pI, myoglobin in this case, while removing other proteins with acidic or basic pIs. For the suppression of electro-osmotic flow (EOF) and protein adsorption, the capillary inner wall was modified with a zwitterionic phospholipid bilayer coating. A preconcentration factor of up to 1700 was obtained for a 1 microg/mL solution of myoglobin. The preconcentration was completed in approximately 20 min. Several sample introduction conditions were presented to accommodate sample volume from one to a few hundreds of microliters. The final volume of the preconcentrated sample band was estimated to be approximately 5 nL.
Formation of Phosphopeptide-metal Ion Complexes in Liquid Chromatography/electrospray Mass Spectrometry and Their Influence on Phosphopeptide Detection
Rapid Communications in Mass Spectrometry : RCM. 2005 | Pubmed ID: 16136520
Despite major advances in mass spectrometry, the detection of phosphopeptides by liquid chromatography with electrospray mass spectrometry (LC/ES-MS) still remains very challenging in proteomics analysis. Phosphopeptides do not protonate efficiently due to the presence of one or more acidic phosphate groups, making their detection difficult. However, other mechanisms also contribute to the difficulties in phosphopeptide analysis by LC/ES-MS. We report here on one such undocumented problem: the formation of phosphopeptide-metal ion complexes during LC/ES-MS. It is demonstrated that both synthetic phosphopeptides and phosphopeptides from bovine beta-casein and alpha-casein form phosphopeptide-metal ion complexes containing iron and aluminum ions, resulting in a dramatic decrease in signal intensity of the protonated phosphopeptides. The interaction of phosphopeptides with metal ions on the surface of the C18 stationary phase is also shown to alter their chromatographic behavior on reversed-phase columns such that the phosphopeptides, especially multiply phosphorylated peptides, become strongly retained and very difficult to elute. The sources of iron and aluminum are from the solvents, stainless steel, glassware and C18 material. It was also found that, upon addition of EDTA, the formation of the phosphopeptide-metal ion complex is diminished, and the phosphopeptides that did not elute from the LC column can now be detected efficiently as protonated molecules. The sensitivity of detection was greatly increased such that a tetra-phosphorylated peptide, RELEELNVPGEIVEpSLpSpSpSEESITR from the tryptic digestion of bovine beta-casein, was detected at a limit of detection of 25 fmol, which is 400 times lower than without EDTA.
Analytical Chemistry. Sep, 2005 | Pubmed ID: 16159144
The analysis of phosphopeptides by mass spectrometry (MS) is one of the most challenging tasks in proteomics. This is due to the lower isoelectric point (pI) of phosphopeptides, which leads to inefficient sample ionization in MS, particularly when competing with other peptides. The problem is compounded by the typical low abundance of phosphopeptides in biological samples. We describe here a simple nonsorptive method to isolate phosphopeptides based on their pI. A voltage is applied to selectively migrate the phosphopeptides into a capillary, which are negatively charged at acidic pH. The selectively sampled fraction is directly deposited onto MALDI sample target in nanoliter volumes (7-35 nL) for highly sensitive MS detection. No significant sample loss is evident in this procedure; hence, the MS was able to detect the isolated phosphopeptides at trace quantity. In this case, attomole-level detection limit is achieved for synthetic phosphopeptides (nM concentration and nL volume), from a mixture containing other peptides at up to 1 million times higher in concentration. Selective sampling was also applied to the tryptic digest of beta- and alpha-caseins to reveal the multiple phosphorylated peptides at the low-femtomole level using MALDI MS. Knowledge of pI based on the rejection/injection of peptides was found to be useful in peak assignment. To confirm the sequence of the selectively sampled peptides, fraction collection was performed for offline ESI MS/MS analysis.
Characterization of Discontinuous Buffer Junctions Using PH Indicators in Capillary Electrophoresis for Protein Preconcentration
Journal of Chromatography. A. Nov, 2006 | Pubmed ID: 17022988
An effective sample preconcentration technique for proteins and peptides was recently developed using capillary electrophoresis (CE) with discontinuous buffers [C.A. Nesbitt, J.T.-M. Lo, K.K.-C. Yeung, J. Chromatogr. A 1073 (2005) 175]. Two buffers of different pH created a junction to trap the sample molecules at their isoelectric points and resulted in over 1000-fold preconcentration for myoglobin within 30 min. To study the formation of pH junctions in CE, a pH indicator, bromothymol blue, is used in this work to reveal the pH changes at the discontinuous buffer boundary. Bromothymol blue (BTB) exhibits a drastic change in its visible absorption spectrum (300-600 nm) going from the acidic to basic pH conditions, and is therefore ideal for visualizing the changes in pH at the junctions created by various buffer combinations. Preconcentration of myoglobin was performed in discontinuous buffers containing BTB. Major differences in the BTB absorption profiles were identified from buffer systems that differ significantly in preconcentration performance, which in turn, allowed for the identification of ideal buffers for sample preconcentration. Up to 2000-fold preconcentrations of myoglobin were achieved in the buffer systems studied in this work. In addition, the role of the electroosmotic flow (EOF) on the preconcentration performance was investigated. A low EOF was found to be desirable, as the pH junction could stay longer in the capillary for accumulation of proteins. The pH junction also displayed characteristics to resist bandbroadening. Potential laminar flow resulted from the mismatched residual EOFs under the two pH conditions within the discontinuous buffers appeared to have minimal effect on the preconcentration. In fact, external applied pressure can be used to control the migration of the pH junction without compromising the protein preconcentration.
Selective Sampling of Multiply Phosphorylated Peptides by Capillary Electrophoresis for Electrospray Ionization Mass Spectrometry Analysis
Journal of Chromatography. A. Jul, 2007 | Pubmed ID: 17188697
The ionization of phosphorylated peptides in positive ion mode mass spectrometry is generally less efficient compared with the ionization of their non-phosphorylated counterparts. This can make phosphopeptides much more difficult to detect. One way to enhance the detection of phosphorylated proteins and peptides is by selectively isolating these species. Current approaches of phosphopeptide isolation are based on the favorable interactions of phosphate groups with immobilized metals. While these methods can be effective in the extraction, they can lead to incomplete sample recovery, particularly for the most strongly bound multiply phosphorylated components. A non-sorptive method of phosphopeptide isolation using capillary electrophoresis (CE) was recently reported [Zhang et al., Anal. Chem. 77 (2005) 6078]. The relatively low isoelectric points of phosphopeptides cause them to remain anionic at acidic sample pH. Hence, they can be selectively injected into the capillary by an applied field after the electroosmotic flow (EOF) is suppressed. The technique was previously coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In this work, the exploitation of selective sampling in conjugation with electrospray ionization mass spectrometry (ESI-MS) is presented. The transition was not immediately straightforward. A number of major alterations were necessary for ESI interfacing. These adaptations include the choice of a suitable capillary coating for EOF control and the incorporation of organic solvent for efficient ESI. As expected, selective injection of phosphopeptides greatly enhanced the sensitivity of their detection in ESI-MS, particularly for the multiply phosphorylated species that were traditionally most problematic. Furthermore, an electrophoretic separation subsequent to the selective injection of the phosphopeptides was performed prior to analysis by ESI-MS. This allowed us to resolve the multiply phosphorylated peptides present in the samples, predominantly based on the number of phosphorylation sites on the peptides.
An Integrated Procedure of Selective Injection, Sample Stacking and Fractionation of Phosphopeptides for MALDI MS Analysis
Analytica Chimica Acta. Jan, 2007 | Pubmed ID: 17386453
Protein phosphorylation is one of the most important post-translational modifications (PTM), however, the detection of phosphorylation in proteins using mass spectrometry (MS) remains challenging. This is because many phosphorylated proteins are only present in low abundance, and the ionization of the phosphorylated components in MS is very inefficient compared to the non-phosphorylated counterparts. Recently, we have reported a selective injection technique that can separate phosphopeptides from non-phosphorylated peptides due to the differences in their isoelectric points (pI) . Phosphorylated peptides from alpha-casein were clearly observed at low femtomole level using MALDI MS. In this work, further developments on selective injection of phosphopeptides are presented to enhance its capability in handling higher sample complexity. The approach is to integrate selective injection with a sample stacking technique used in capillary electrophoresis to enrich the sample concentration, followed by electrophoresis to fractionate the components in preparation for MALDI MS analysis. The effectiveness of the selective injection and stacking was evaluated quantitatively using a synthetic phosphopeptide as sample, with an enrichment factor of up to 600 being recorded. Next, a tryptic digest of alpha-casein was used to evaluate the separation and fractionation of peptides for MALDI MS analysis. The elution order of phosphopeptides essentially followed the order of decreasing number of phosphates on the peptides. Finally, to illustrate the applicability, the integrated procedure was applied to evaluate the phosphorylation of a highly phosphorylated protein, osteopontin. Up to 41 phosphopeptides were observed, which allowed us to examine the phosphorylation of all 29 possible sites previously reported . A high level of heterogeneity in the phosphorylation of OPN was evident by the multiple-forms of variable phosphorylation detected for a large number of peptides.
Nanoliter-volume Protein Enrichment, Tryptic Digestion, and Partial Separation Based on Isoelectric Points by CE for MALDI Mass Spectral Analysis
Electrophoresis. Jan, 2008 | Pubmed ID: 18081201
Sequence-specific proteolysis is an important part of protein identification by MS. Digestion of protein is commonly performed in-solution, in sample vials with volumes ranging from milli- to microliters. When digestion is performed with a sample volume below 1 microL, handling of solution and potential sample loss via adsorption become significant issues. In this report, a proof of concept for the digestion of a small volume protein solution inside a capillary was demonstrated using a discontinuous buffer system previously studied (Nesbitt, C. A., et al. J. Chromatogr. A 2005, 1073, 175-180). Upon voltage application, a pH junction was created by the discontinuous buffer. Using myoglobin as an example, the protein molecules were enriched at the junction with an estimated volume of a few nanoliters. A protease, trypsin, was then introduced to myoglobin at the junction by coenrichment to induce in-capillary digestion. The voltage application was then suspended to provide the necessary time (2 h) for the proteolysis to proceed. When completed, voltage application was resumed, and the discontinuous buffer reconcentrated the peptides formed from digestion. Importantly, the refocused peptides appeared to roughly elute according to their pIs, resulting in a partial separation. Direct sample deposition from capillary was performed to facilitate mass spectral analysis by MALDI. The partial separation, according to pI, offered the potential benefits of MALDI MS signal enhancement and provided supplementary pI information for peptide identity assignment.
Recent Applications of Capillary Electrophoresis-mass Spectrometry (CE-MS): CE Performing Functions Beyond Separation
Analytica Chimica Acta. Oct, 2008 | Pubmed ID: 18790124
Capillary electrophoresis is one of the separation tools commonly used in conjugation with mass spectrometry. Its primary purpose is to resolve the components in a sample mixture prior to mass spectral identification. Moreover, an increasing number of applications reported in the literature involve the use of CE for additional purposes, such as sample preparation and derivatization, and the study of biochemical properties. This review provides an overview on the various roles of CE beyond that of a simple separation tool. While the scope focuses on the area of interest rather than a predefined time period, the majority of the references highlighted were initially published within the past five years.
In-capillary Protein Enrichment and Removal of Nonbuffering Salts Using Capillary Electrophoresis with Discontinuous Buffers
Analytical Chemistry. Nov, 2008 | Pubmed ID: 18925747
Salt is abundant in biological samples and can cause problems in capillary electrophoresis (CE) due to excessive Joule heating and electrodispersion. Desalting with solid phase minibeds is currently most compatible with the small sample volumes of CE. They are however difficult to prepare and suffer from poor bed-to-bed reproducibility. Alternatively, enrichment of proteins and peptides was developed using CE, by trapping them at their isoelectric points with a discontinuous buffer of mismatched pH. Ionic salts, such as sodium chloride, do not possess isoelectric points and therefore are not retained by the discontinuous buffer. In this work, the removal of ionic salt during protein enrichment using CE with discontinuous buffers was investigated. Nonbuffering ions were found to electromigrate through the pH junction without disrupting the enrichment process and were eventually removed from the capillary. Mass spectral data obtained from the enriched and desalted sample confirmed a significant signal enhancement. Finally, a strong acid was introduced to remove the pH junction and thus facilitated a subsequent capillary zone electrophoresis separation. An integrated procedure of enrichment, desalting, and separation was demonstrated on a mixture of three protein standards.
In-capillary Enrichment, Proteolysis and Separation Using Capillary Electrophoresis with Discontinuous Buffers: Application on Proteins with Moderately Acidic and Basic Isoelectric Points
The Analyst. Jan, 2009 | Pubmed ID: 19082176
Advances in mass spectrometry and capillary-format separation continue to improve the sensitivity of protein analysis. Of equal importance is the miniaturization of sample pretreatment such as enrichment and proteolysis. In a previous report (Nesbitt et al., Electrophoresis, 2008, 29, 466-474), nanoliter-volume protein enrichment, tryptic digestion, and partial separation was demonstrated in capillary electrophoresis followed by MALDI mass spectral analysis. A discontinuous buffer system, consisting of ammonium (pH 10) and acetate (pH 4), was used to create a pH junction inside the capillary, trapping a protein with a neutral isoelectric point, myoglobin (pI 7.2). Moreover, co-enrichment of myoglobin with trypsin led to an in-capillary digestion. In this paper, the ability of this discontinuous buffer system to perform similar in-capillary sample pretreatment on proteins with moderately acidic and basic pI was studied and reported. Lentil lectin (pI 8.6) and a multi-phosphorylated protein, beta-casein (pI 5.1), were selected as model proteins. In addition to the previously shown tryptic digestion, proteolysis with endoproteinase Asp-N was also performed. Digestion of these acidic and basic pI proteins produced a few peptides with extreme pI values lying outside the trapping range of the discontinuous buffer. An alteration in the peptide trapping procedure was made to accommodate these analytes. Offline MALDI mass spectral analysis confirmed the presence of the expected peptides. The presented miniaturized sample pretreatment methodology was proven to be applicable on proteins with a moderately wide range of pI. Flexibility in the choice of protease was also evident.
Sphingomyelins As Semi-permanent Capillary Coatings for Protein Separations in CE and Off-line Analysis with MALDI-MS
Electrophoresis. May, 2009 | Pubmed ID: 19360777
Phosphatidylcholine (PC) is one of the major phospholipids that make up the biological cell membrane. It was previously reported to form capillary inner wall coatings for CE. The zwitterionic head group of PC produced a neutral net-charged bilayer, which was found effective in preventing wall adsorption of both cationic and anionic proteins [J. M. Cunliffe et al. Anal. Chem. 2002, 74, 776-783]. Another major membrane phospholipid that possesses a zwitterionic head group is sphingomyelin (SM). In this work, the novel characterization of SM on its effectiveness in capillary coating formation for CE separations of proteins and peptides was presented. Similar properties were observed between PC and SM, including their effects on the EOF, peak efficiencies, and migration time reproducibilities. SM appeared to be more readily soluble in aqueous solutions, and it was found equally effective as PC in facilitating protein separation. The main difference observed was their performances in delivering a peptide mixture for off-line analysis with MALDI-MS. Superior sample recovery was evident from the capillary coated with SM compared with that with PC. The number of peptides identified from a 1 ng/microL myglobin tryptic digest sample increased from 5 to 16 (42 and 69%, respectively in protein sequence coverage).
Removal of Sample Background Buffering Ions and Myoglobin Enrichment Via a PH Junction Created by Discontinuous Buffers in Capillary Electrophoresis
Journal of Chromatography. A. Aug, 2011 | Pubmed ID: 21762923
Traditional CE sample stacking is ineffective for samples containing a high concentration of salt and/or buffer. We recently reported the use of a discontinuous buffer system for protein enrichment that was applicable to samples containing millimolar concentrations of salt. In this paper, the technique was investigated for samples containing unwanted buffering ions, including TRIS, MES, and phosphate, which are commonly used in biological sample preparation. Using myoglobin as a model protein, the results demonstrated that background buffering ions can be effectively removed or separated from the enriched protein. The key is to use either the acid or the base of the discontinuous buffers to adjust the pH of the sample, such that the net charge of the unwanted buffering ions is near-zero. The successful isolation and enrichment of myoglobin from up to 100 mM TRIS and 50 mM MES was demonstrated. The enrichment factors remained at approximately 200. Removal of phosphate was more challenging because its net charge was anionic in both the acid and the base of the discontinuous buffers. The enrichment was only achievable up to 30 mM of sodium phosphate, the enrichment factors observed were significantly lower, below 50, and the process was delayed due to the higher ionic strength resulted from phosphate. The migration of phosphate during enrichment was studied using a UV-absorbing analogue, phenyl phosphate. In addition, Simul 5.0 was used to simulate the discontinuous buffers in the absence and presence of TRIS and phosphate. The stimulated TRIS and phosphate concentration profiles were generally in agreement with the experimental results. The simulation also provided a better understanding on the effect of phosphate on the formation of the pH junction.
The Analyst. Oct, 2012 | Pubmed ID: 22919699
Capillary electrophoresis (CE) is not only an effective separation technique, but can also serve as a sample preparation tool for enrichment and purification at sub-microliter sample volumes. Our approach is based on the use of a discontinuous buffer system consisting of an acid and a base (acetate and ammonium). Proteins and/or peptides with isoelectric points between the pH values of these two buffers will become stacked at the neutralization reaction boundary (NRB). To understand the mechanism of the NRB formation and the electrophoretic migration of various ions during the enrichment, we performed experiments using myoglobin and mesityl oxide to reveal the ion migration patterns at the buffer junction, and utilized Simul 5 to computer simulate the process. The simulated results closely resembled the experimental data, and together, they effectively revealed the characteristics of the discontinuous buffers. Importantly, the discovery allowed the manipulation of NRB behaviours by controlling the discontinuous buffer composition. To illustrate this, the removal of urea as an unwanted background molecule from the enriched protein sample was achieved based on the acquired information.
Journal of Agricultural and Food Chemistry. Apr, 2014 | Pubmed ID: 24697626
Agricultural crop residues can be converted through thermochemical pyrolysis to bio-oil, a sustainable source of biofuel and biochemicals. The pyrolysis bio-oil is known to contain many chemicals, some of which have insecticidal activity and can be a potential source of value-added pest control products. Brassicacae crops, cabbage, broccoli, and mustards, contain glucosinolates and isocyanates, compounds with recognized anti-herbivore activity. In Canada, canola Brassica napus straw is available from over 6 000 000 ha and mustard Brassica carinata and Brassica juncea straw is available from 200 000 ha. The straw can be converted by microbial lignocellulosic enzymes as a substrate for bioethanol production but can also be converted to bio-oil by thermochemical means. Straw from all three species was pyrolyzed, and the insecticidal components in the bio-oil were isolated by bioassay-guided solvent fractionation. Of particular interest were the mustard straw bio-oil aqueous fractions with insecticidal and feeding repellent activity to Colorado potato beetle larvae. Aqueous fractions further analyzed for active compounds were found not to contain many of the undesirable phenol compounds, which were previously found in other bio-oils seen in the dichloromethane (DCM) and ethyl acetate (EA) solvent phases of the present study. Identified within the most polar fractions were hexadecanoic and octadecanoic fatty acids, indicating that separation of these compounds during bio-oil production may provide a source of effective insecticidal compounds.
Increased Expression of Simple Ganglioside Species GM2 and GM3 Detected by MALDI Imaging Mass Spectrometry in a Combined Rat Model of Aβ Toxicity and Stroke
PloS One. 2015 | Pubmed ID: 26086081
The aging brain is often characterized by the presence of multiple comorbidities resulting in synergistic damaging effects in the brain as demonstrated through the interaction of Alzheimer's disease (AD) and stroke. Gangliosides, a family of membrane lipids enriched in the central nervous system, may have a mechanistic role in mediating the brain's response to injury as their expression is altered in a number of disease and injury states. Matrix-Assisted Laser Desorption Ionization (MALDI) Imaging Mass Spectrometry (IMS) was used to study the expression of A-series ganglioside species GD1a, GM1, GM2, and GM3 to determine alteration of their expression profiles in the presence of beta-amyloid (Aβ) toxicity in addition to ischemic injury. To model a stroke, rats received a unilateral striatal injection of endothelin-1 (ET-1) (stroke alone group). To model Aβ toxicity, rats received intracerebralventricular (i.c.v.) injections of the toxic 25-35 fragment of the Aβ peptide (Aβ alone group). To model the combination of Aβ toxicity with stroke, rats received both the unilateral ET-1 injection and the bilateral icv injections of Aβ25-35 (combined Aβ/ET-1 group). By 3 d, a significant increase in the simple ganglioside species GM2 was observed in the ischemic brain region of rats who received a stroke (ET-1), with or without Aβ. By 21 d, GM2 levels only remained elevated in the combined Aβ/ET-1 group. GM3 levels however demonstrated a different pattern of expression. By 3 d GM3 was elevated in the ischemic brain region only in the combined Aβ/ET-1 group. By 21 d, GM3 was elevated in the ischemic brain region in both stroke alone and Aβ/ET-1 groups. Overall, results indicate that the accumulation of simple ganglioside species GM2 and GM3 may be indicative of a mechanism of interaction between AD and stroke.
Identification of Six New Alternaria Sulfoconjugated Metabolites by High-resolution Neutral Loss Filtering
Rapid Communications in Mass Spectrometry : RCM. Oct, 2015 | Pubmed ID: 26331931
Many species of Alternaria damage important agricultural crops, including small grains and tomatoes. These fungi can produce a variety of secondary metabolites, some of which are toxic to humans and animals. Interest in screening for conjugated or 'modified' mycotoxins has increased because of their tendency to evade traditional analytical screening methods. Two sulfoconjugated Alternaria toxins have been reported and the potential exists for many more.
Differential Anatomical Expression of Ganglioside GM1 Species Containing D18:1 or D20:1 Sphingosine Detected by MALDI Imaging Mass Spectrometry in Mature Rat Brain
Frontiers in Neuroanatomy. 2015 | Pubmed ID: 26648849
GM1 ganglioside plays a role in essential neuronal processes, including differentiation, survival, and signaling. Yet, little is known about GM1 species with different sphingosine bases, such as the most abundant species containing 18 carbon atoms in the sphingosine chain (GM1d18:1), and the less abundant containing 20 carbon atoms (GM1d20:1). While absent in the early fetal brain, GM1d20:1 continues to increase throughout pre- and postnatal development and into old age, raising questions about the functional relevance of the GM1d18:1 to GM1d20:1 ratio. Matrix-assisted laser desorption/ionization imaging mass spectrometry is a novel technology that allows differentiation between these two GM1 species and quantification of their expression within an anatomical context. Using this technology, we find GM1d18:1/d20:1 expression ratios are highly specific to defined anatomical brain regions in adult rats. Thus, the ratio was significantly different among different thalamic nuclei and between the corpus callosum and internal capsule. Differential GM1d18:1/GM1d20:1 ratios measured in hippocampal subregions in rat brain complement previous studies conducted in mice. Across layers of the sensory cortex, opposing expression gradients were found for GM1d18:1 and GM1d20:1. Superficial layers demonstrated lower GM1d18:1 and higher GM1d20:1 signal than other layers, while in deep layers GM1d18:1 expression was relatively high and GM1d20:1 expression low. By far the highest GM1d18:1/d20:1 ratio was found in the amygdala. Differential expression of GM1 with d18:1- or d20:1-sphingosine bases in the adult rat brain suggests tight regulation of expression and points toward a distinct functional relevance for each of these GM1 species in neuronal processes.
Journal of Agricultural and Food Chemistry. Feb, 2016 | Pubmed ID: 26837797
Several Aspergillus species produce ochratoxin A (OTA) and/or fumonisins on wine and table grapes. The relevant species and their mycotoxins have been investigated in a number of wine-producing regions around the world; however, similar data have not been reported for Canadian vineyards. A multiyear survey of black Aspergilli in Niagara, ON, vineyards was conducted to determine the diversity of species present and to assess the risk of OTA and fumonisin contamination of wine grapes from this region. From 2012 to 2014, 253 black Aspergilli were isolated from soil samples and the fruits of 10 varieties of grapes. The isolates were identified by DNA sequencing: Aspergillus welwitschiae (43%), Aspergillus uvarum (32%), Aspergillus brasiliensis (11%), Aspergillus tubingensis (9%), and Aspergillus niger (4%). Aspergillus carbonarius, the primary OTA producer on grapes in other parts of the world, was isolated only once, and this is the first report for it in Canada. All 10 A. niger strains produced fumonisins, but, in contrast, only 26% of the 109 A. welwitschiae isolates were producers, and no strains of either species produced OTA. Grape samples were analyzed for OTA and fumonisins from sites where strains with mycotoxigenic potential were isolated. Fumonisin B2 (FB2) was detected in 7 of 22 (32%) of these grape samples in the 1-15 ppb range, but no OTA was detected. Additionally, the recently reported nonaminated fumonisins were detected in 3 of 22 grape samples. These results suggest that fumonisin-producing Aspergilli can occur in Ontario vineyards but, at present, the risk of contamination of grapes appears low. The risk of OTA contamination in Niagara wine is also low because of the low prevalence of A. carbonarius.
Miniaturized Digestion and Extraction of Surface Proteins from Candida Albicans Following Treatment with Histatin 5 for Mass Spectrometry Analysis
International Journal of Proteomics. 2016 | Pubmed ID: 28044106
A common approach to isolate surface proteins from fungal and bacterial cells is to perform a proteolytic cleavage of proteins on the surface of intact cells suspended in solution. This paper describes miniaturization of this technique, in which cells are adhered on glass surfaces, and all sample treatments are conducted at μL volumes. Specifically, Candida albicans cells were attached onto HSA-coated glass slides. By depositing the appropriate reagent solutions on the adhered cells, we successfully performed cell washing, treatment with antifugal peptide, Histatin 5, and a proteolysis on intact cells with trypsin. The resulting peptides were subsequently analysed by mass spectrometry. In general, the data obtained was similar to that collected with suspended cells in much larger sample volumes. However, our miniaturized workflow offers the benefit of greatly reducing the consumption of cells and reagents.