Increased expression of the ER membrane acetyl-CoA transporter AT-1 can cause an autism-like phenotype in mice.
The import of acetyl-CoA into the lumen of the endoplasmic reticulum (ER) by AT-1/SLC33A1 regulates Nε-lysine acetylation of ER-resident and -transiting proteins. Specifically, lysine acetylation within the ER appears to influence the efficiency of the secretory pathway by affecting ER-mediated quality control. Mutations or duplications in AT-1/SLC33A1 have been linked to diseases such as familial spastic paraplegia, developmental delay with premature death, and autism spectrum disorder with intellectual disability. In this study, we generated an AT-1 Tg mouse model that selectively overexpresses human AT-1 in neurons. These animals demonstrate cognitive deficits, autistic-like social behavior, aberrations in synaptic plasticity, an increased number of dendritic spines and branches, and widespread proteomic changes. We also found that AT-1 activity regulates acetyl-CoA flux, causing epigenetic modulation of the histone epitope H3K27 and mitochondrial adaptation. In conclusion, our results indicate that increased expression of AT-1 can cause an autistic-like phenotype by affecting key neuronal metabolic pathways.
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging (MSI) is a powerful tool to map the spatial distribution of biomolecules on tissue sections. Recent developments of hybrid MS instruments allow combination of different types of data acquisition by various mass analyzers into a single MSI analysis, which reduces experimental time and sample consumptions. Here, using the well-characterized crustacean nervous system as a test-bed, we explore the utility of high resolution and accurate mass (HRAM) MALDI Orbitrap platform for enhanced in situ characterization of the neuropeptidome with improved chemical information. Specifically, we report on a multiplex-MSI method, which combines HRAM MSI with data dependent acquisition (DDA) tandem MS analysis in a single experiment. This method enables simultaneous mapping of neuropeptide distribution, sequence validation and novel neuropeptide discovery in crustacean neuronal tissues. To enhance the dynamic range and efficiency of in situ DDA, we introduced a novel approach of fractionating full m/z range into several sub-mass ranges and embedding the setup using the multiplex-DDA-MSI scan events to generate pseudo fractionation before MS/MS scans. The division of entire m/z into multiple segments of m/z subranges for MS interrogation greatly decreased the complexity of molecular species from tissue samples and the heterogeneity of the distribution and variation of intensities of m/z peaks. By carefully optimizing the experimental conditions such as the dynamic exclusion, the multiplex-DDA-MSI approach demonstrates better performance with broader precursor coverage, less biased MS/MS scans towards high abundance molecules and improved quality of tandem mass spectra for low intensity molecular species.
MALDI MS imaging; multiplex MS imaging; HRAM; neuropeptide; peptidomics; crustacean nervous system
Environmental fluctuations, such as salinity, impose serious challenges to marine animal survival. Neuropeptides, signaling molecules involved in the regulation process, and the dynamic changes of their full complement in the stress response have yet to be investigated. Here, a MALDI-MS-based stable isotope labeling quantitation strategy was used to investigate the relationship between neuropeptide expression and adaptability of Carcinus maenas to various salinity levels, including high (60 p.p.t.) and low (0 p.p.t.) salinity, in both the crustacean pericardial organ (PO) and brain. Moreover, a high salinity stress time course study was conducted. MS imaging (MSI) of neuropeptide localization in Carcinus maenas PO was also performed. As a result of salinity stress, multiple neuropeptide families exhibited changes in their relative abundances, including RFamides (e.g. APQGNFLRFamide), RYamides (e.g. SSFRVGGSRYamide), B type-allatostatins (AST-B) (e.g. VPNDWAHFRGSWamide), and orcokinins (e.g. NFDEIDRSSFGFV). The MSI data revealed distribution differences in several neuropeptides (e.g. SGFYANRYamide) between color morphs, but salinity stress appeared to not have a major effect on the localization of the neuropeptides.
crustacean; mass spectrometry imaging; neuropeptides; isotopic labeling; salinity stress
Decapod crustaceans are important animal models for neurobiologists due to their relatively simple nervous systems with well-defined neural circuits and extensive neuromodulation by a diverse set of signaling peptides. However, biochemical characterization of these endogenous neuropeptides is often challenging due to limited sequence information about these neuropeptide genes and the encoded preprohormones. By taking advantage of sequence homology in neuropeptides observed in related species using a home-built crustacean neuropeptide database, we developed a semi-automated sequencing strategy to characterize the neuropeptidome of Panulirus interruptus, an important aquaculture species, with few known neuropeptide preprohormone sequences. Our streamlined process searched the high mass accuracy and high-resolution data acquired on a LTQ-Orbitrap with a flexible algorithm in ProSight that allows for sequence discrepancy from reported sequences in our database, resulting in the detection of 32 neuropeptides, including 19 novel ones. We further improved the overall coverage to 51 neuropeptides with our multidimensional platform that employed multiple analytical techniques including dimethylation-assisted fragmentation, de novo sequencing using nanoliquid chromatog raphy-electrospray ionization-quadrupole-time-of-flight (nanoLC–ESI–Q-TOF), direct tissue analysis, and mass spectrometry imaging on matrix-assisted laser desorption/ionization (MALDI)-TOF/TOF. The high discovery rate from this unsequenced model organism demonstrated the utility of our neuropeptide discovery pipeline and highlighted the advantage of utilizing multiple sequencing strategies. Collectively, our study expands the catalog of crustacean neuropeptides and more importantly presents an approach that can be adapted to exploring neuropeptidome from species that possess limited sequence information.
neuropeptide; Panulirus interruptus; de novo sequencing; ProSight; high-resolution and high-accuracy mass spectrometry; dimethylation-assisted fragmentation; mass spectrometric imaging; peptidomics
Mass spectrometry (MS) coupled to sample preparation and separation techniques has become a primary tool for proteomics studies. However, due to sample complexity, it is often challenging to achieve fast and efficient sample preparation prior to MS analysis. In recent decades, monolithic materials have been developed not only as chromatographic media, but also as efficient solid supports for immobilizing multiple types of affinity reagents. Herein, the N-acryloxysuccinimide-co-acrylamide-co-N,N'-methylenebisacrylamide (NAS-AAm-Bis) monolith was fabricated within silanized 200 μm i.d. fused-silica capillaries and was used as an immobilized enzyme reactor (IMER). The column was conjugated with trypsin/Lys-C and Lys-N enzymes to allow enzymatic digestions to occur while protein mixture was loaded onto the IMER column followed by MS-based proteomics analysis. Similar MS signal and protein sequence coverage were observed using protein standard bovine serum albumin (BSA) compared to in-solution digestion. Furthermore, mouse serum, yeast, and human cell lysate samples were also subjected to enzymatic digestion by both IMER (in seconds to minutes) and conventional in solution digestion (overnight) for comparison in large-scale proteomics studies. Comparable protein identification results obtained by the two methods highlighted the potential of employing NAS-based IMER column for fast and highly efficient sample preparation for MS analysis in proteomics studies.
monolithic column; proteomics; protein identification; ESI-Orbitrap-MS; digestion; IMER; MALDI-MS; database search
Lower urinary tract symptoms (LUTS) are a range of irritative or obstructive symptoms that commonly afflict aging population. The diagnosis is mostly based on patient-reported symptoms, and current medication often fails to completely eliminate these symptoms. There is a pressing need for objective non-invasive approaches to measure symptoms and understand disease mechanisms. We developed an in-depth workflow combining urine metabolomics analysis and machine learning bioinformatics to characterize metabolic alterations and support objective diagnosis of LUTS. Machine learning feature selection and statistical tests were combined to identify candidate biomarkers, which were statistically validated with leave-one-patient-out cross-validation and absolutely quantified by selected reaction monitoring assay. Receiver operating characteristic analysis showed highly-accurate prediction power of candidate biomarkers to stratify patients into disease or non-diseased categories. The key metabolites and pathways may be possibly correlated with smooth muscle tone changes, increased collagen content, and inflammation, which have been identified as potential contributors to urinary dysfunction in humans and rodents. Periurethral tissue staining revealed a significant increase in collagen content and tissue stiffness in men with LUTS. Together, our study provides the first characterization and validation of LUTS urinary metabolites and pathways to support the future development of a urine-based diagnostic test for LUTS.
Neuropeptides represent one of the largest classes of signaling molecules used by nervous systems to regulate a wide range of physiological processes. Over the past several years, mass spectrometry (MS)-based strategies have revolutionized the discovery of neuropeptides in numerous model organisms, especially in decapod crustaceans. Here, we focus our discussion on recent advances in the use of MS-based techniques to map neuropeptides in spatial domain and monitoring their dynamic changes in temporal domain. These MS-enabled investigations provide valuable information about the distribution, secretion and potential function of neuropeptides with high molecular specificity and sensitivity. In situ MS imaging and in vivo microdialysis are highlighted as key technologies for probing spatio-temporal dynamics of neuropeptides in the crustacean nervous system. This review summarizes the latest advancement in MS-based methodologies for neuropeptide analysis including typical workflow and sample preparation strategies as well as major neuropeptide families discovered in decapod crustaceans.
neuropeptide; mass spectrometry; mass spectrometric imaging; crustacean; microdialysis; peptidomics
Relative quantification of proteins via their enzymatically digested peptide products determines disease biomarker candidate lists in discovery studies. Isobaric label-based strategies using TMT and iTRAQ allow for up to 10 samples to be multiplexed in one experiment, but their expense limits their use. The demand for cost-effective tagging reagents capable of multiplexing many samples led us to develop an 8-plex version of our isobaric labeling reagent, DiLeu.
The original 4-plex DiLeu reagent was extended to an 8-plex set by coupling isotopic variants of dimethylated leucine to an alanine balance group designed to offset the increasing mass of the label’s reporter group. Tryptic peptides from a single protein digest, a protein mixture digest, and Saccharomyces cerevisiae lysate digest were labeled with 8-plex DiLeu and analyzed via nanoLC-MS2 on a Q-Exactive Orbitrap mass spectrometer. Characteristics of 8-plex DiLeu-labeled peptides, including quantitative accuracy and fragmentation, were examined.
An 8-plex set of DiLeu reagents with 1 Da-spaced reporters was synthesized at a yield of 36%. The average cost to label eight 100 μg peptide samples was calculated to be approximately $15. Normalized collision energy tests on the Q-Exactive revealed that a higher-energy collisional dissociation value of 27 generated the optimum number of high-quality spectral matches. Relative quantification of DiLeu-labeled peptides yielded normalized median ratios accurate to within 12% of their expected values.
Cost-effective 8-plex DiLeu reagents can be synthesized and applied to relative peptide and protein quantification. These labels increase the multiplexing capacity of our previous 4-plex implementation without requiring high-resolution instrumentation to resolve reporter ion signals.
isobaric labels; 8-plex DiLeu; relative quantification; multiplexing
Neuropeptides (NPs), a unique and highly important class of signaling molecules across the animal kingdom, have been extensively characterized in the neuronal tissues of various crustaceans. Because many NPs are released into circulating fluid (hemolymph) and travel to distant sites in order to exhibit physiological effects, it is important to measure the secretion of these NPs from living animals. In this study, we report on extensive characterization of NPs released in the crab Cancer borealis by utilizing in vivo microdialysis to sample NPs from the hemolymph. We determined the necessary duration for collection of microdialysis samples, enabling more comprehensive identification of NP content while maintaining the temporal resolution of sampling. Analysis of in vivo microdialysates using a hybrid quadrupole-Orbitrap™ Q-Exactive mass spectrometer revealed that more than 50 neuropeptides from 9 peptide families—including the allatostatin, RFamide, orcokinin, tachykinin-related peptide and RYamide families–were released into the circulatory system. The presence of these peptides both in neuronal tissues as well as in hemolymph indicates their putative hormonal roles, a finding that merits further investigation. Preliminary quantitative measurement of these identified NPs suggested several potential candidates that may be associated with the circadian rhythm in Cancer borealis.
neuropeptide; secretion; mass spectrometry; microdialysis; crustacean; hemolymph; in vivo sampling
Due to the significance of protein phosphorylation in various biological processes and signaling events, new analytical techniques for enhanced phosphoproteomics have been rapidly introduced in recent years. The combinatorial use of the phospho-specific enrichment techniques and prefractionation methods prior to MS analysis enables comprehensive profiling of the phosphoproteome and facilitates deciphering the critical roles that phosphorylation plays in signaling pathways in various biological systems. This review places special emphasis on the recent five-year (2009–2013) advances for enrichment and separation techniques that have been utilized for phosphopeptides prior to MS analysis.
Enrichment; separation; phosphopeptides; phosphoproteomics; mass spectrometry
Hypoxia has adverse effects on renal development. This study was the first to test hypoxia-induced renal autophagy in rat fetuses.
Pregnant rats were exposed to hypoxia or normoxia during pregnancy and fetal kidneys were collected at gestation day 21.
Fetal kidney weight and ratio of kidney–body weight were reduced. Histological analysis showed enlargement in Bowman space and wider space between interstitia in the kidneys of fetus exposed to hypoxia. Fetal renal B-cell lymphoma 2 (BCL-2) was decreased accompanied with higher 2′-deoxyuridine 5′-triphosphate nick end-labeling staining and unchanged soluble FAS in the hypoxia group. Hypoxia increased autophagic structures, including autophagosomes and autolysosomes, in fetal kidneys and increased renal APG5L. There was an increase in renal LC3-II, Beclin 1, p-S6, hypoxia inducible factor 1α (HIF-1a), and ratio of LC3-II–LC3-I and a decrease in P62, protein kinase B (AKT), and phosphorylated AKT in the hypoxia group. Both renal mammalian target of rapamycin (mTOR) and Beclin 1 signaling were upregulated.
Hypoxia-affected fetal renal development was associated with renal apoptosis and Beclin 1 signaling-mediated autophagy.
hypoxia; autophagy; rat fetus; kidney
Tandem mass spectrometry (MS/MS)-based relative quantification by isobaric labeling is a useful technique to compare different metabolic expression levels in biological systems. For the first time, we have labeled primary and secondary amine-containing small molecules using 4-plex isobaric N,N-dimethyl-leucine (DiLeu) to perform relative quantification. Good labeling efficiency and quantification accuracy were demonstrated with a mixture of 12 metabolite standards including amino acids and small molecule neurotransmitters. Labeling amine-containing metabolites with DiLeu reagents also enabled the separation of polar metabolites by nanoRPLC and improved the detection sensitivity by CE-ESI-MS. The 4-plex DiLeu labeling technique combined with LC-MS/MS and CE-MS/MS platforms were applied to profile and quantify amine-containing metabolites in mouse urine. The variability of concentrations of identified metabolites in urine samples from different mouse individuals was illustrated by the ratios of reporter ion intensities acquired from online data-dependent analysis.
Serotonin neurons located in the raphe nucleus of the hindbrain have crucial roles in regulating brain functions and have been implicated in various psychiatric disorders. Yet functional human serotonin neurons are not available for in vitro studies. Through manipulation of the WNT pathway, we demonstrate efficient differentiation of human pluripotent stem cells (hPSCs) to cells resembling central serotonin neurons, primarily those located in the rhombomeric segments 2–3 of the rostral raphe, which participate in high-order brain functions. The serotonin neurons express a series of molecules essential for serotonergic development, including tryptophan hydroxylase 2, exhibit typical electrophysiological properties and release serotonin in an activity-dependent manner. When treated with the FDA-approved drugs tramadol and escitalopram oxalate, they release or uptake serotonin in a dose- and time-dependent manner, suggesting the utility of these cells for the evaluation of drug candidates.
Absolute quantification of protein targets using liquid chromatography-mass spectrometry (LC-MS) is a key component of candidate biomarker validation. One popular method combines multiple reaction monitoring (MRM) using a triple quadrupole instrument with stable isotope-labeled standards (SIS) for absolute quantification (AQUA). LC-MRM AQUA assays are sensitive and specific, but they are also expensive due to the cost of synthesizing stable isotope peptide standards. While the chemical modification approach using Mass Differential Tags for Relative and Absolute Quantification (mTRAQ) represents a more economical approach when quantifying large numbers of peptides, these reagents are costly and still suffer from lower throughput because only two concentration values per peptide can be obtained in a single LC-MS run. Here, we have developed and applied a set of five novel mass difference reagents, isotopic N,N-dimethyl leucine (iDiLeu). These labels contain an amine reactive group, triazine ester, are cost effective due to their synthetic simplicity, and have increased throughput compared to previous LC-MS quantification methods by allowing construction of a four-point standard curve in one run. iDiLeu-labeled peptides show remarkably similar retention time shifts, slightly lower energy thresholds for higher-energy collisional dissociation (HCD) fragmentation, and high quantification accuracy for trypsin-digested protein samples (median errors <15%). By spiking in an iDiLeu-labeled neuropeptide, allatostatin, into mouse urine matrix, two quantification methods are validated. The first uses one labeled peptide as an internal standard to normalize labeled peptide peak areas across runs (<19% error) while the second enables standard curve creation and analyte quantification in one run (<8% error).
Protein/peptide quantification; iDiLeu; DiLeu; absolute quantification; isotopic labeling; standard curve; dimethylated amino acid
Legumes have developed the unique ability to establish a symbiotic relationship with soil bacteria known as rhizobia. This interaction results in the formation of root nodules in which rhizobia thrive and reduce atmospheric dinitrogen into plant-usable ammonium through biological nitrogen fixation (BNF). Due to the availability of genetic information for both of the symbiotic partners, the Medicago truncatula–Sinorhizobium meliloti association is an excellent model for examining the BNF process. Although metabolites are important in this symbiotic association, few studies have investigated the array of metabolites that influence this process. Of these studies, most target only a few specific metabolites, the roles of which are either well known or are part of a well-characterized metabolic pathway. Here, we used a multifaceted mass spectrometric (MS) approach to detect and identify the key metabolites that are present during BNF using the Medicago truncatula–Sinorhizobium meliloti association as the model system. High mass accuracy and high resolution matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) Orbitrap instruments were used in this study and provide complementary results for more in-depth characterization of the nitrogen-fixation process. We used well-characterized plant and bacterial mutants to highlight differences between the metabolites that are present in functional vs. non-functional nodules. Our study highlights the benefits of using a combination of mass spectrometric techniques to detect differences in metabolite composition and the distributions of these metabolites in plant biology.
Nitrogen Fixation; Medicago truncatula; Metabolites; MALDI; Orbitrap; Mass Spectrometry; Imaging; Q-Exactive
Food consumption is an important
behavior that is regulated by
an intricate array of neuropeptides (NPs). Although many feeding-related
NPs have been identified in mammals, precise mechanisms are unclear
and difficult to study in mammals, as current methods are not highly
multiplexed and require extensive a priori knowledge about analytes.
New advances in data-independent acquisition (DIA) MS/MS and the open-source
quantification software Skyline have opened up the possibility to
identify hundreds of compounds and quantify them from a single DIA
MS/MS run. An untargeted DIA MSE quantification method
using Skyline software for multiplexed, discovery-driven quantification
was developed and found to produce linear calibration curves for peptides
at physiologically relevant concentrations using a protein digest
as internal standard. By using this method, preliminary relative quantification
of the crab Cancer borealis neuropeptidome (<2
kDa, 137 peptides from 18 families) was possible in microdialysates
from 8 replicate feeding experiments. Of these NPs, 55 were detected
with an average mass error below 10 ppm. The time-resolved profiles
of relative concentration changes for 6 are shown, and there is great
potential for the use of this method in future experiments to aid
in correlation of NP changes with behavior. This work presents an
unbiased approach to winnowing candidate NPs related to a behavior
of interest in a functionally relevant manner, and demonstrates the
success of such a UPLC-MSE quantification method using
the open source software Skyline.
Data-independent acquisition; mass spectrometry; neuropeptide; microdialysis; feeding; quantification
The MALDI-LTQ-Orbitrap XL mass spectrometer is a high performance instrument capable of high resolution and accurate mass (HRAM) measurements. The maximum m/z of 4000 precludes the MALDI analysis of proteins without generating multiply charged ions. Herein, we present the study of HRAM laserspray ionization mass spectrometry (MS) with MS/MS and MS imaging capabilities using 2-nitrophloroglucinol (2-NPG) as matrix on a MALDI-LTQ-Orbitrap XL mass spectrometer. The optimized conditions for multiply charged ion production have been determined and applied to tissue profiling and imaging. Biomolecules as large as 15 kDa have been detected with up to five positive charges at 100K mass resolution (at m/z 400). More importantly, MS/MS and protein identification on multiply charged precursor ions from both standards and tissue samples have been achieved for the first time with an intermediate-pressure source. The initial results reported in this study highlight potential utilities of laserspray ionization MS analysis for simultaneous in situ protein identification, visualization and characterization from complex tissue samples on a commercially available HRAM MALDI MS system.
Ion mobility (IM) is a gas-phase electrophoretic method that separates ions according to charge and ion-neutral collision cross-section (CCS). Herein, we attempt to apply a travelling wave (TW) IM polyalanine calibration method to shotgun proteomics and create a large peptide CCS database. Mass spectrometry methods that utilize IM, such as HDMSE, often use high transmission voltages for sensitive analysis. However, polyalanine calibration has only been demonstrated with low voltage transmission used to prevent gas-phase activation. If polyalanine ions change conformation under higher transmission voltages used for HDMSE, the calibration may no longer be valid. Thus, we aimed to characterize the accuracy of calibration and CCS measurement under high transmission voltages on a TW IM instrument using the polyalanine calibration method and found that the additional error was not significant. We also evaluated the potential error introduced by liquid chromatography (LC)-HDMSE analysis, and found it to be insignificant as well, validating the calibration method. Finally, we demonstrated the utility of building a large-population peptide CCS database by investigating the effects of terminal lysine position, via LysC or LysN digestion, on the formation of two structural sub-families formed by triply charged ions.
The crustacean stomatogastric nervous system (STNS) is a classic experimental model to derive basic knowledge about neuron functions and how they coordinate with each other to generate neural circuits. To investigate the components of the neuromodulators and how they are distributed in such system is essential to understand the underlying mechanism. In this study, in situ mass spectrometry based techniques were employed to fulfill this goal.
Offline high performance liquid chromatography (HPLC) separation was coupled with matrix-assisted laser desorption/ionization time of flight/ time of flight (MALDI-TOF/TOF) to analyze the neuropeptides in the stomatogastric ganglion (STG) tissue extract from the Jonah crab Cancer borealis. Direct tissue analysis was also employed to investigate the neuropeptides presented in the STNS. MALDI imaging was also applied to map the localization of multiple neuropeptide families in the STG and the upstream nerve.
57 neuropeptides were detected from a single desheathed STG using direct tissue analysis, and they were from 11 different neuropeptide families, including FaRP, AST-A, AST-B, etc. Differential neuropeptide profiles from three different types of ganglia and two types of nerve fiber tissues from the STNS were documented. The direct tissue analysis was shown better for studying neuropeptides from small neural organs like the STG as compared to the large scale HPLC-MALDI analysis. MALDI images were also acquired to study the distribution of neuropeptides in the STG.
In this study, the components and distribution of neuropeptides have been analyzed in the STNS from C. borealis using direct tissue profiling and MALDI imaging. The results show that the direct tissue analysis of desheathed neural tissues can provide higher sensitivity for neuropeptide study compared to large scale HPLC-MALDI analysis of pooled tissues. The results are valuable for understanding the functions of neuropeptides in neural network generation.
Cancer borealis; direct tissue analysis; MALDI-TOF/TOF; mass spectrometric imaging (MSI); neuropeptide; stomatogastric nervous system (STNS)
isobaric tags (e.g., tandem mass tags (TMT) and isobaric
tags for relative and absolute quantification (iTRAQ)) are a valuable
tool for high-throughput mass spectrometry based quantitative proteomics.
We have developed our own multiplex isobaric tags, DiLeu, that feature
quantitative performance on par with commercial offerings but can
be readily synthesized in-house as a cost-effective alternative. In
this work, we achieve a 3-fold increase in the multiplexing capacity
of the DiLeu reagent without increasing structural complexity by exploiting
mass defects that arise from selective incorporation of 13C, 15N, and 2H stable isotopes in the reporter
group. The inclusion of eight new reporter isotopologues that differ
in mass from the existing four reporters by intervals of 6 mDa yields
a 12-plex isobaric set that preserves the synthetic simplicity and
quantitative performance of the original implementation. We show that
the new reporter variants can be baseline-resolved in high-resolution
higher-energy C-trap dissociation (HCD) spectra, and we demonstrate
accurate 12-plex quantitation of a DiLeu-labeled Saccharomyces
cerevisiae lysate digest via high-resolution nano
liquid chromatography–tandem mass spectrometry (nanoLC–MS2) analysis on an Orbitrap Elite mass spectrometer.
matrix application technique is critical to the success of
a matrix-assisted laser desorption/ionization (MALDI) experiment.
This work presents a systematic study aiming to evaluate three different
matrix application techniques for MALDI mass spectrometric imaging
(MSI) of endogenous metabolites from legume plant, Medicago
truncatula, root nodules. Airbrush, automatic sprayer, and
sublimation matrix application methods were optimized individually
for detection of metabolites in the positive ionization mode exploiting
the two most widely used MALDI matrices, 2,5-dihydroxybenzoic acid
(DHB) and α-cyano-4-hydroxycinnamic acid (CHCA). Analytical
reproducibility and analyte diffusion were examined and compared side-by-side
for each method. When using DHB, the optimized method developed for
the automatic matrix sprayer system resulted in approximately double
the number of metabolites detected when compared to sublimation and
airbrush. The automatic sprayer method also showed more reproducible
results and less analyte diffusion than the airbrush method. Sublimation
matrix deposition yielded high spatial resolution and reproducibility
but fewer analytes in the higher m/z range (500–1000 m/z). When
the samples were placed in a humidity chamber after sublimation, there
was enhanced detection of higher mass metabolites but increased analyte
diffusion in the lower mass range. When using CHCA, the optimized
automatic sprayer method and humidified sublimation method resulted
in double the number of metabolites detected compared to standard
Temperature changes influence the
reaction rates of all biological
processes, which can pose dramatic challenges to cold-blooded organisms,
and the capability to adapt to temperature fluctuations is crucial
for the survival of these animals. In order to understand the roles
that neuropeptides play in the temperature stress response, we employed
a mass spectrometry-based approach to investigate the neuropeptide
changes associated with acute temperature elevation in three neural
tissues from the Jonah crab Cancer borealis. At high temperature, members from two neuropeptide families, including
RFamide and RYamide, were observed to be significantly reduced in
one of the neuroendocrine structures, the pericardial organ, while
several orcokinin peptides were detected to be decreased in another
major neuroendocrine organ, the sinus gland. These results implicate
that the observed neuropeptides may be involved with temperature perturbation
response via hormonal regulation. Furthermore, a temperature stress
marker peptide with the primary sequence of SFRRMGGKAQ (m/z 1137.7) was detected and de novo sequenced in
the circulating fluid (hemolymph) from animals under thermal perturbation.
Cancer borealis; neuropeptide; temperature change; dimethyl labeling; quantitative
peptidomics; mass spectrometry; pericardial organ; sinus gland; MALDI-TOF/TOF; MALDI-FTMS
The relative quantification of proteins using liquid chromatography mass spectrometry (LC-MS) has allowed researchers to compile lists of potential disease markers. These complex quantitative workflows often include isobaric labeling of enzymatically-produced peptides to analyze their relative abundances across multiple samples in a single LC-MS run. Recent efforts by our lab have provided scientists with cost-effective alternatives to expensive commercial labels. Although the quantitative performance of these dimethyl leucine (DiLeu) labels has been reported using known ratios of complex protein and peptide standards, their potential in large-scale proteomics studies using a clinically relevant system has never been investigated. Our work rectifies this oversight by implementing 4-plex DiLeu to quantify proteins in the urine of aging human males who suffer from lower urinary tract symptoms (LUTS). Protein abundances in 25 LUTS and 15 control patients were compared, revealing that of the 836 proteins quantified, 50 were found to be differentially expressed (>20% change) and statistically significant (p-value <0.05). Gene ontology (GO) analysis of the differentiated proteins showed that many were involved in inflammatory responses and implicated in fibrosis. While confirmation of individual protein abundance changes would be required to verify protein expression, this study represents the first report using the custom isobaric label, 4-plex DiLeu, to quantify protein abundances in a clinically relevant system.
Hypoxia during pregnancy could affect development of fetuses as well as cardiovascular systems in the offspring. This study was the first to demonstrate the influence and related mechanisms of prenatal hypoxia (PH) on renal interlobar arteries (RIA) in the 5-month-old male rat offspring. Following chronic hypoxia during pregnancy, phenylephrine induced significantly higher pressor responses and greater vasoconstrictions in the offspring. Nitric oxide mediated vessel relaxation was altered in the RIA. Phenylephrine-stimulated free intracellular calcium was significantly higher in the RIA of the PH group. The activity and expression of L-type calcium channel (Cav1.2), not T-type calcium channel (Cav3.2), was up-regulated. The whole-cell currents of calcium channels and the currents of Cav1.2 were increased compared with the control. In addition, the whole-cell K+ currents were decreased in the offspring exposed to prenatal hypoxia. Activity of large-conductance Ca2+-activated K+ channels and the expression of MaxiKα was decreased in the PH group. The results provide new information regarding the influence of prenatal hypoxia on the development of the renal vascular system, and possible underlying cellular and ion channel mechanisms involved.
Isobaric tandem mass tags are an attractive alternative to mass difference tags and label free approaches for quantitative proteomics due to the high degree of multiplexing that can be performed with their implementation. A drawback of tandem mass tags are that the co-isolation and co-fragmentation of labeled peptide precursors can result in chimeric MS/MS spectra that can underestimate the fold-change expression of each peptide. Two methods (QuantMode and MS3) have addressed this concern for ion trap and orbitrap instruments, but there is still a need to solve this problem for quadrupole time-of-flight (Q-TOF) instruments. Ion mobility (IM) separations coupled to Q-TOF instruments have the potential to mitigate MS/MS spectra chimeracy since IM-MS has the ability to separate ions based on charge, m/z, and collision cross section (CCS). This work presents results that showcase the power of IM-MS to improve tandem mass tag peptide quantitation accuracy by resolving co-isolated differently charged and same charged peptides prior to MS/MS fragmentation.
tandem mass tags; Di-Leu (dimethylated leucine); ion mobility; quantitation; Q-TOF