scholarly journals Affinity-Bead Assisted Mass Spectrometry (Affi-BAMS): A Multiplexed Microarray Platform for Targeted Proteomics

2020 ◽  
Vol 21 (6) ◽  
pp. 2016
Author(s):  
Ghaith M. Hamza ◽  
Vladislav B. Bergo ◽  
Sergey Mamaev ◽  
Don M. Wojchowski ◽  
Paul Toran ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Binding Capacity ◽  
Microarray Platform ◽  
Magnetic Bead ◽  
Maldi Ms ◽  
Ionization Mass ◽  
Affinity Capture ◽  
Intact Proteins ◽  
Single Bead ◽  
Multiple Samples

The ability to quantitatively probe diverse panels of proteins and their post-translational modifications (PTMs) across multiple samples would aid a broad spectrum of biological, biochemical and pharmacological studies. We report a novel, microarray analytical technology that combines immuno-affinity capture with Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS), which is capable of supporting highly multiplexed, targeted proteomic assays. Termed “Affinity-Bead Assisted Mass Spectrometry” (Affi-BAMS), this LC-free technology enables development of highly specific and customizable assay panels for simultaneous profiling of multiple proteins and PTMs. While affinity beads have been used previously in combination with MS, the Affi-BAMS workflow uses enrichment on a single bead that contains one type of antibody, generally capturing a single analyte (protein or PTM) while having enough binding capacity to enable quantification within approximately 3 orders of magnitude. The multiplexing capability is achieved by combining Affi-BAMS beads with different protein specificities. To enable screening of bead-captured analytes by MS, we further developed a novel method of performing spatially localized elution of targets from individual beads arrayed on a microscope slide. The resulting arrays of micro spots contain highly concentrated analytes localized within 0.5 mm diameter spots that can be directly measured using MALDI MS. While both intact proteins and protein fragments can be monitored by Affi-BAMS, we initially focused on applying this technology for bottom-up proteomics to enable screening of hundreds of samples per day by combining the robust magnetic bead-based workflow with the high throughput nature of MALDI MS acquisition. To demonstrate the variety of applications and robustness of Affi-BAMS, several studies are presented that focus on the response of 4EBP1, RPS6, ERK1/ERK2, mTOR, Histone H3 and C-MET to stimuli including rapamycin, H2O2, EPO, SU11274, Staurosporine and Vorinostat.

scholarly journals MALDIViz: A Comprehensive Informatics Tool for MALDI-MS Data Visualization and Analysis

2017 ◽  
Vol 22 (10) ◽  
pp. 1246-1252 ◽  
Author(s):  
Kishore Kumar Jagadeesan ◽  
Simon Ekström
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Web Application ◽  
Low Cost ◽  
Maldi Ms ◽  
Ionization Mass ◽  
Label Free ◽  
Label Free Detection ◽  
R Shiny

Recently, mass spectrometry (MS) has emerged as an important tool for high-throughput screening (HTS) providing a direct and label-free detection method, complementing traditional fluorescent and colorimetric methodologies. Among the various MS techniques used for HTS, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) provides many of the characteristics required for high-throughput analyses, such as low cost, speed, and automation. However, visualization and analysis of the large datasets generated by HTS MALDI-MS can pose significant challenges, especially for multiparametric experiments. The datasets can be generated fast, and the complexity of the experimental data (e.g., screening many different sorbent phases, the sorbent mass, and the load, wash, and elution conditions) makes manual data analysis difficult. To address these challenges, a comprehensive informatics tool called MALDIViz was developed. This tool is an R-Shiny-based web application, accessible independently of the operating system and without the need to install any program locally. It has been designed to facilitate easy analysis and visualization of MALDI-MS datasets, comparison of multiplex experiments, and export of the analysis results to high-quality images.

scholarly journals Rapid Alterations in Cerebral White Matter Lipid Profiles After Ischemic-Reperfusion Brain Injury in Fetal Sheep as Demonstrated by MALDI-Mass Spectrometry

2019 ◽  
Vol 22 (4) ◽  
pp. 344-355 ◽  
Author(s):  
Gina M Gallucci ◽  
Ming Tong ◽  
Xiaodi Chen ◽  
Barbara S Stonestreet ◽  
Amy Lin ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
White Matter ◽  
Lipid Composition ◽  
Lipid Profiles ◽  
Maldi Ms ◽  
Cerebral White Matter ◽  
Ionization Mass ◽  
Myelin Lipid ◽  
Fetal Sheep ◽  
Bilateral Carotid

Background Perinatal ischemia-reperfusion (I/R) injury of cerebral white matter causes long-term cognitive and motor disabilities in children. I/R damages or kills highly metabolic immature oligodendroglia via oxidative stress, excitotoxicity, inflammation, and mitochondrial dysfunction, impairing their capacity to generate and maintain mature myelin. However, the consequences of I/R on myelin lipid composition have not been characterized. Objective This study utilized matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to assess alterations in cerebral supraventricular white matter myelin lipid profiles in a fetal sheep model of perinatal I/R. Methods Fetal sheep (127 days gestation) were studied after 30 minutes of bilateral carotid artery occlusion followed by 4 (n = 5), 24 (n = 7), 48 (n = 3), or 72 (n = 5) hours of reperfusion, or sham treatment (n = 5). White matter lipids were analyzed by negative ion mode MALDI-MS. Results Striking I/R-associated shifts in phospholipid and sphingolipid expression occurred over the 72-hour time course with most responses detected within 4 hours of reperfusion and progressing at the 48- and 72-hour points. I/R decreased expression of phosphatidic acid and phosphatidylethanol amine and increased phosphatidylinositol, sulfatide, and lactosylceramide. Conclusions Cerebral I/R in mid-gestation fetal sheep causes rapid shifts in white matter myelin lipid composition that may reflect injury, proliferation, or recovery of immature oligodendroglia.

Identification of a Crystalline Drug Metabolite in Tissue Sections using Microscopy and Maldi-MS

2000 ◽  
Vol 6 (S2) ◽  
pp. 1002-1003
Author(s):  
J. Fagerland ◽  
L. Miesbauer ◽  
R. Burton ◽  
F. Seiler ◽  
J. Neilly ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Metabolic Pathways ◽  
Renal Toxicity ◽  
Polarized Light ◽  
Parent Drug ◽  
Maldi Ms ◽  
Drug Candidate ◽  
Ionization Mass ◽  
X Ray ◽  
Tissue Sections

Development of a potential drug candidate was discontinued when rats treated with the compound developed severe renal toxicity after one week of daily dosing. Tubular degeneration in the kidney was accompanied by deposition of large amounts of crystalline material, which was also present in spleen and pancreas (Fig. 1). It was presumed that the crystals were precipitated parent drug or one of its metabolites. Using microscopy and mass spectrometry, it was confirmed that the crystals were indeed derived from the drug and not endogenously formed; in addition, the precise metabolite that had precipitated in the tissues was identified, providing clues to the metabolic pathways involved.Frozen sections of phosphate-buffered formaldehyde-fixed kidney, pancreas, and spleen were evaluated by polarized light microscopy (PLM), scanning electron microscopy (SEM) with energy dispersive x-ray spectroscopy (EDXS), and matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS). For reference, crystals of the trifluoroacetate salt of the parent drug were also analyzed using these methods.

scholarly journals Application of dyes as doping agents in MALDI-MS matrices for the signal enhancement of proteins

RSC Advances ◽  
2017 ◽  
Vol 7 (11) ◽  
pp. 6598-6604
Author(s):  
Arslan Ali ◽  
Najia Shahid ◽  
Syed Ghulam Musharraf
Keyword(s):  
Mass Spectrometry ◽  
Laser Desorption ◽  
Maldi Ms ◽  
Ionization Mass Spectrometry ◽  
Ionization Mass ◽  
Laser Desorption Ionization ◽  
As Doping ◽  
Desorption Ionization ◽  
Matrix Assisted Laser ◽  
Matrix Assisted Laser Desorption

Dyes can significantly influence the ionization of proteins, when used as dopants in conventional matrices used for Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

scholarly journals Influences of Blood Sample Processing on Low–Molecular-Weight Proteome Identified by Surface-Enhanced Laser Desorption/Ionization Mass Spectrometry

2005 ◽  
Vol 51 (9) ◽  
pp. 1637-1649 ◽  
Author(s):  
Rosamonde E Banks ◽  
Anthea J Stanley ◽  
David A Cairns ◽  
Jennifer H Barrett ◽  
Paul Clarke ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Laser Desorption ◽  
Ionization Mass ◽  
Plasma Samples ◽  
Laser Desorption Ionization ◽  
Serum Samples ◽  
Affinity Capture ◽  
Desorption Ionization ◽  
Elapsed Time ◽  
Surface Enhanced

Abstract Background: Profiling approaches in proteomics, such as surface-enhanced laser desorption/ionization (SELDI) mass spectrometry, are used in disease marker discovery. The aim of this study was to investigate the potential influence of selected preanalytical factors on the results obtained. Methods: Plasma samples anticoagulated with EDTA, citrate, or heparin, and serum samples from healthy volunteers were profiled by SELDI on CM10, immobilized metal affinity capture (IMAC) array with copper, and H50 chip surfaces. Using linear mixed-effects models, we examined the influence of elapsed time between venipuncture and sample separation (immediate to 24 h) and the type of serum tube used (Greiner Vacuette activator, gel serum separator, or plain tubes). We analyzed purified platelets, as well as platelet-poor and platelet-rich plasma samples treated with calcium and/or thrombin to determine the platelet contribution, directly or via the clotting process, to the profiles generated. We then used cluster analysis to identify samples with similar peak profiles. Results: Different plasma types and sera could be distinguished on the basis of cluster analyses of their spectral profiles. Elapsed time between venipuncture and separation of plasma and serum from blood samples altered the profiles obtained, particularly for serum samples and particularly on IMAC chips. The type of serum collection tube also affected the profiles because of differences in clotting time. In vitro manipulation of platelets revealed that specific peaks in IMAC profiles of serum appeared to be derived directly from platelets. Several other peaks, including some of those exhibiting time-dependent changes, arose during the clotting process. Conclusion: Preanalytical variables, such as sample handling, can markedly influence results.

scholarly journals Study on titanium dioxide nanoparticles as MALDI MS matrix for the determination of lipids in the brain

2021 ◽  
Vol 10 (1) ◽  
pp. 700-710
Author(s):  
Congnan Peng ◽  
Qian Zhang ◽  
Jian-an Liu ◽  
Zhen-peng Wang ◽  
Zhen-wen Zhao ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Titanium Dioxide ◽  
Titanium Dioxide Nanoparticles ◽  
Brain Homogenate ◽  
Maldi Ms ◽  
Absolute Ethanol ◽  
Future Research ◽  
Ionization Mass ◽  
The Matrix

Abstract The structures of lipids are diverse, and thus, lipids show various biological functions. Systematic determination of lipids in organisms has always been a concern. In this paper, a methodology on the matrix-assisted laser desorption ionization mass spectrometry (MALDI MS), with titanium dioxide nanoparticles (TiO2 NPs) as the matrix, was studied for lipid determination. The results showed that the following conditions were preferable in the determination of small-molecule lipids (such as hypoxanthine, guanosine, uridine, and cytidine), lipid standards (such as GC, GM, TG, phosphatidylethanolamine, phosphatidylcholine, and ceramide), and mixed lipids (extracted from brain homogenate with methanol alone and with the B&D method): TiO2 NPs as the matrix, absolute ethanol as the solvent, 1 mg of TiO2 NPs dispersed in 1 mL of absolute ethanol as the matrix solution, NaCl as the ionization reagent, and positive mass spectrometry (MS) as the mode. Modified TiO2 NP as a new matrix for MALDI MS will be a future research direction; in addition, the characteristics of TiO2 NPs make it a potential matrix for imaging MS.

Functionalized Titanium Oxide Nanowire Substrate for Surface-Assisted Laser Desorption/ionization Imaging Mass Spectrometry

2019 ◽  
Author(s):  
Ewelina P. Dutkiewicz ◽  
Han-Jung Lee ◽  
Cheng-Chih Hsu ◽  
Yu-Liang Yang
Keyword(s):  
Mass Spectrometry ◽  
Secondary Metabolites ◽  
Imaging Mass Spectrometry ◽  
Detection Limits ◽  
Laser Desorption ◽  
Maldi Ms ◽  
Ionization Mass ◽  
Laser Desorption Ionization ◽  
Powerful Technique ◽  
Desorption Ionization

Imaging mass spectrometry (IMS) is a powerful technique that enables analysis of various molecular species at a high spatial resolution with low detection limits. In contrast to the standard matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) approach, surface-assisted laser desorption/ionization (SALDI) is more effective in the detection of small molecules due to the absence of interfering background signals in low <i>m/z</i> ranges. We developed a functionalized TiO<sub>2</sub> nanowire as a solid substrate for IMS of low-molecular-weight species in biological specimens. We prepared TiO<sub>2</sub> nanowires using the inexpensive modified hydrothermal process and subsequently functionalized it chemically with various silane analogs to overcome the problem of superhydrophilicity of the substrate. Chemical modification changed the selectivity of imprinting of samples deposited on the surface of the plate and thus improved the detection limits. Due to the enhanced performance, the functionalized TiO<sub>2</sub> nanowire substrate could be successfully used for imaging of complex native samples. We applied our new substrate to image distribution of the secondary metabolites in (1) petal of the medicinal plant <i>Catharanthus roseus</i> and (2) microbial co-culture of <i>Burkholderia</i> <i>cenocepacia </i>869T2 vs <i>Phellinus noxius</i>. We observed that secondary metabolites are distributed heterogeneously in a petal, which is consistent with previous results reported for the <i>C. roseus</i> plant leaf and stem. We verified the semi-quantitative capabilities of the imprinting/imaging approach by comparing results using standard LC-MS analysis of the plant extracts. Several bacteria-related metabolites produced by <i>B</i>. <i>cenocepacia</i> 869T2 in presence of <i>P. noxius</i>, which were unable to be detected by MALDI-MS approach, were revealed by our newly developed approach. This suggested that the functionalized TiO<sub>2</sub> nanowire substrates-based SALDI is a powerful technique complementary to MALDI-MS.

Plasma Lipidomic Fingerprinting to Distinguish among Hepatitis C-related Hepatocellular Carcinoma, Liver Cirrhosis, and Chronic Hepatitis C using MALDI-TOF Mass Spectrometry: a Pilot Study

2015 ◽  
Vol 24 (1) ◽  
pp. 43-49 ◽  
Author(s):  
Ana Maria Passos-Castilho ◽  
Edson Lo Turco ◽  
Maria Lúcia Ferraz ◽  
Carla Matos ◽  
Ivonete Silva ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Hepatocellular Carcinoma ◽  
Liver Cirrhosis ◽  
Chronic Hepatitis ◽  
Hepatitis C ◽  
Chronic Hepatitis C ◽  
Predictive Accuracy ◽  
Maldi Ms ◽  
Charge Ratio ◽  
Ionization Mass

Background & Aims: Hepatitis C (HC) is a major cause of hepatocellular carcinoma (HCC), and a late diagnosis is the main factor for the poor survival of patients. There is an urgent need for identifying sensitive and specific biomarkers for HCC diagnosis. In the present study, plasma lipid patterns of patients with HC-HCC, HC-liver cirrhosis (LC), and chronic HC (CHC) were assessed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).Methods. Plasma samples of 25 patients with HC-HCC, 15 patients with HC-LC, and 25 patients with CHC were evaluated by MALDI-MS using a Q-ToF premier (Synapt) mass spectrometer (Waters, Manchester, UK) equipped with a 200-Hz solid-state laser in the mass range between m/z (mass-to-charge ratio) of 700-1200.Results. A total of 2205 ions were initially obtained and 7 ions (m/z) were highlighted as corresponding to the most important lipids to differentiate HCC patients from LC and CHC patients. The specific lipidomic expression signature generated resulted in an overall predictive accuracy of 93% of HC-HCC and HC-LC, and 100% of HC-HCC and CHC. The 7-peak algorithm distinguished HCC from LC with a sensitivity of 96% and a specificity of 87%, and HCC from CHC with both sensitivity and specificity of 100%.Conclusion. MALDI-MS-specific signature peaks accurately distinguished patients with HC-HCC from those with HC-LC and CHC. The results indicate the potential of MALDI-MS and the selected peaks to improve HCC surveillance in patients with viral C cirrhosis and chronic hepatitis C.

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