scholarly journals Online μSEC2-nRPLC-MS for improved sensitivity of intact protein detection of IEF separated non-human primate cerebrospinal fluid proteins

2021 ◽  
Author(s):  
Erika N Cline ◽  
Caroline Alvarez ◽  
Steven M Patrie
Keyword(s):  
Mass Spectrometry ◽  
Cerebrospinal Fluid ◽  
Modular Design ◽  
Dynamic Range ◽  
Protein Detection ◽  
Size Exclusion ◽  
Mass Spectrometry Analysis ◽  
Intact Protein ◽  
Chemical Complexity ◽  
Target Mass

Proteoform-resolved information, obtained by top-down (TD) ″intact protein″ proteomics, is expected to contribute substantially to the understanding of molecular pathogenic mechanisms and in turn, identify novel therapeutic and diagnostic targets. However, the robustness of mass spectrometry analysis of intact proteins in complex mixtures is hindered by high dynamic range in protein concentration and mass, protein instability, and the chemical complexity of biological samples. Here, we describe an evolutionary step for intact protein investigations through the online implementation of tandem microflow size exclusion chromatography with nanoflow reversed-phase liquid chromatography and mass spectrometry (μSEC2-nRPLC-MS). Online serial high- and low-pass SEC filtration overcomes the aforementioned hurdles to intact proteomic analysis through automated sample desalting/cleanup and enrichment of target mass ranges prior to nRPLC-MS analysis. The coupling of μSEC to nRPLC is achieved through a novel injection volume control (IVC) strategy of inserting protein trap columns pre- and post-μSEC columns to enable injection of dilute samples in high volumes without loss of sensitivity or resolution. Critical characteristics of the approach are tested via rigorous investigations on samples of varied complexity and chemical background. Application to cerebrospinal fluid (CSF) samples pre-fractionated by OFFGEL isoelectric focusing demonstrates that the platform drastically increases the number of intact mass tags detected within the target mass range (< 30 kDa) in comparison to one-dimensional nRPLC-MS starting from approximately 100x less CSF than previous studies. Furthermore, the modular design of the μSEC2-nRPLC-MS platform is robust and promises significant flexibility for large-scale TDMS analysis of diverse samples either directly or in concert with other multidimensional fractionation steps.

scholarly journals A preliminary study of Dihydroorotase from Methanococcus jannaschii

2014 ◽  
Vol 70 (a1) ◽  
pp. C481-C481
Author(s):  
Aditya Singh ◽  
Michael Colaneri ◽  
Jacqueline Vitali
Keyword(s):  
Mass Spectrometry ◽  
De Novo ◽  
Hydrophobic Interaction Chromatography ◽  
Size Exclusion ◽  
Mass Spectrometry Analysis ◽  
Yale University ◽  
Spectrometry Analysis ◽  
Bacteria And Fungi ◽  
Physics Department ◽  
Research Institute

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamoyl-L-aspartate to form L-dihydroorotate in the third step of de novo pyrimidine biosynthesis. It is a Zinc metalloenzyme and a member of the aminohydrolase superfamily. There are two classes of the enzyme. Class I, typically ~45 kDa, is found in higher organisms, bacteria and yeast. Class II, typically ~38 kDa, is found in bacteria and fungi. Some organisms have multiple DHOase sequences. The M. jannaschii pyrC gene coding for DHOase was subcloned and expressed in E. coli. Protein purification consisted of ammonium sulfate precipitation, heat treatment at 850C, and phenyl-sepharose hydrophobic interaction chromatography. The protein was confirmed in the SDS gel using Liquid Chromatography-Mass Spectrometry (Proteomics Laboratory, Lerner Research Institute, Cleveland, OH). Size Exclusion Chromatography-Laser Light Scattering (Keck Biotechnology Laboratory, Yale University, New Haven, CT) indicated that the protein is a monomer in solution with a molecular weight of 47 kDa. A model constructed with the I-TASSER server (Zhang, 2008) suggested that the binding site contains only one Zn ion per monomer coordinated by the conserved His56, His58 and Asp302. Asp146 is further away and does not coordinate with the Zn ion. According to the mass spectrometry analysis, the protein does not contain a carboxylated lysine. Our progress on this study will be presented. Acknowledgements: We thank Dr. Belinda Willard (Lerner Research Institute) for the LC-MS and Dr. Ewa Folta-Stogniew (Yale University) for the SEC- LS analysis. The presentation was supported in part by a graduate faculty travel award and by a contribution from the Physics Department at Cleveland State University.

scholarly journals ProbingN-glycoprotein microheterogeneity by lectin affinity purification-mass spectrometry analysis

2019 ◽  
Vol 10 (19) ◽  
pp. 5146-5155 ◽  
Author(s):  
Di Wu ◽  
Jingwen Li ◽  
Weston B. Struwe ◽  
Carol V. Robinson
Keyword(s):  
Mass Spectrometry ◽  
Protein Level ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Intact Protein ◽  
Spectrometry Analysis ◽  
Lectin Affinity ◽  
Affinity Purification Mass Spectrometry

A lectin affinity purification-mass spectrometry approach to characterize lectin-reactive glycoproteoforms and elucidate lectin specificities at the intact protein level.

Mass spectrometry analysis of redox forms of High-Mobility Group Box-1 Protein in cerebrospinal fluid: initial experience.

2019 ◽  
Vol 88 (3) ◽  
pp. 171-176
Author(s):  
Agata Światły ◽  
Norbert Wąsik ◽  
Joanna Hajduk ◽  
Eliza Matuszewska ◽  
Paweł Dereziński ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cerebrospinal Fluid ◽  
Subarachnoid Hemorrhage ◽  
High Mobility ◽  
High Mobility Group ◽  
Mass Spectrometry Analysis ◽  
Aneurysmal Subarachnoid Haemorrhage ◽  
Top Down ◽  
Spectrometry Analysis ◽  
Redox Forms

Introduction. High-mobility group box 1 (HMGB1) is an alarmin with proinflammatory potential determined by redox status of the cysteines at position 23 and 45. It may also play a role as a biomarker in biological fluids. The aim of this study was the identification of different HMGB1 redox forms in cerebrospinal fluid (CSF) obtained from subarachnoid hemorrhage patients. Material and Methods. 6 CSF samples were collected from aneurysmal subarachnoid haemorrhage patients. Commercially available HMGB1 isoforms served as a positive control. Immunoprecipitation and electrophoretic isolation of HMGB1 protein were performed, then both CSF and control were analyzed using mass spectrometry technique. To distinguish between fully reduced (thiol group at C23 and C45) and disulfide (disulfide bond connecting C23 and C45) HMGB1 forms, top-down sequencing of the spectra was performed. Results. Top-down sequencing analysis allowed to distinguish between HMGB1 isoforms only in commercially available standard without preceding immunoprecipitation and electrophoresis. MALDI spectra differ i.e. on the fully reduced HMGB1 spectrum fragmentation occurs before and beyond C22, which is not present on the disulfide HMGB1 spectrum. Analysis of HMGB1 isolated from CSF obtained from subarachnoid hemorrhage patients gave no results. Conclusions. Top-down sequencing enables to distinguish between redox forms of HMGB1. Electrophoresis and tryptic digestion cannot precede mass spectrometry analysis of redox forms of HMGB1 due to the reduction of disulfide bonds during these processes. Preferred method of isolation of HMGB1 for direct analysis using top-down sequencing mustn’t include protein digestion or degradation.

MosaicNeedles: A tool for multi-omic liquid biopsy sensitivity and specificity enhancement.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e15019-e15019
Author(s):  
Qimin Quan ◽  
Joe Wilkinson ◽  
Joshua Ritchey ◽  
Alaina Kaiser ◽  
John Geanacopoulos ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Single Molecule ◽  
Liquid Biopsy ◽  
Dynamic Range ◽  
Color Change ◽  
Protein Detection ◽  
Target Sequence ◽  
Label Free ◽  
Protein Biomarkers ◽  
Oncology Research

e15019 Background: Liquid biopsy has evolved to be an important method complementary to tissue biopsy. It is not only non-invasive, but also has the potential to detect cancer in its earliest stages and monitor patients in remission. The integration of proteomics into liquid biopsy may transform the molecular diagnostics of cancer and accelerate basic and clinical oncology research. A recent study showed that adding just 8 protein biomarkers to a panel of circulating DNA biomarkers increased the diagnostic accuracy up to 98% sensitivity and 99% specificity. Proteomics also bridges the gaps of functional information lost due to post-transcriptional and post-translational modifications in the genomic approach. However, the proteogenomic approach normally requires the use of multiple different assay technologies and laboratory workflows, including mass spectrometry. Methods: NanoMosaic’s Tessie platform employs a densely integrated nanoneedle sensor array (thus named MosaicNeedles) which can be used to detect both nucleic acids and proteins in a single assay process with reduced workflow complexity, without the need for mass spectrometry. Results: The NanoMosaic platform is a label-free, digital, single molecule counting technology using nanoneedles. It achieves sub-pg/ml (̃fM) level sensitivity with 7 logs of dynamic range. An array of nanoneedles is densely integrated and manufactured with CMOS-compatible nanofabrication processes. Each nanoneedle is a single molecule biosensor that is functionalized with capture probes. The capture probe can be either an antibody for protein detection or an oligonucleotide with a specific target sequence to a DNA fragment, mRNA, or miRNA of interest. The scattering spectrum of each nanoneedle changes when an analyte binds to its surface. At low abundance, analytes that are captured can be quantitated by counting the presence or absence of a color change on each individual nanoneedle in a binary fashion. As an analyte concentration increases the binding events increase accordingly and achieve saturation. In this range, an analog analysis on the spectrum shift will be performed, thus providing a wider dynamic range, up to 7 logs. Ultrahigh level multiplex can be achieved by parallelizing each analyte specific sensing area without loss of sensitivity or dynamic range. A 10,000-plex study can be achieved with a total of 2.5 billion nanoneedles on a 50mm by 50mm consumable. In this consumable, a 2,000-plex proteome and 8,000 cell-free DNA fragments can be detected. Conclusions: In conclusion, a full proteogenomic quantification can be performed on the NanoMosaic platform in one reaction, with higher sensitivity, lower cost and higher throughput than is currently possible by traditional methods. In addition, the high-plexibility of the NanoMosaic platform allows the discovery of new biomarkers across the whole proteome without the need for mass spectrometry.

Non-target mass spectrometry analysis of NDMA precursors in advanced treatment for potable reuse

2018 ◽  
Vol 4 (12) ◽  
pp. 1944-1955 ◽  
Author(s):  
Shannon L. Roback ◽  
Imma Ferrer ◽  
E. Michael Thurman ◽  
Kenneth P. Ishida ◽  
Megan H. Plumlee ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
High Resolution Mass Spectrometry ◽  
Mass Spectrometry Analysis ◽  
Advanced Treatment ◽  
Spectrometry Analysis ◽  
Potable Reuse ◽  
Target Mass ◽  
High Resolution Mass ◽  
Resolution Mass

Non-target high-resolution mass spectrometry was used to track the occurrence and removal of NDMA precursors during advanced treatment for reuse.

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