scholarly journals A streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Pan Fang ◽  
Yanlong Ji ◽  
Ivan Silbern ◽  
Carmen Doebele ◽  
Momchil Ninov ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
Sample Preparation ◽  
Large Scale ◽  
Handling Time ◽  
Database Search ◽  
High Confidence ◽  
Site Specific ◽  
Sample Handling ◽  
Data Processing Tool

Abstract Regulation of protein N-glycosylation is essential in human cells. However, large-scale, accurate, and site-specific quantification of glycosylation is still technically challenging. We here introduce SugarQuant, an integrated mass spectrometry-based pipeline comprising protein aggregation capture (PAC)-based sample preparation, multi-notch MS3 acquisition (Glyco-SPS-MS3) and a data-processing tool (GlycoBinder) that enables confident identification and quantification of intact glycopeptides in complex biological samples. PAC significantly reduces sample-handling time without compromising sensitivity. Glyco-SPS-MS3 combines high-resolution MS2 and MS3 scans, resulting in enhanced reporter signals of isobaric mass tags, improved detection of N-glycopeptide fragments, and lowered interference in multiplexed quantification. GlycoBinder enables streamlined processing of Glyco-SPS-MS3 data, followed by a two-step database search, which increases the identification rates of glycopeptides by 22% compared with conventional strategies. We apply SugarQuant to identify and quantify more than 5,000 unique glycoforms in Burkitt’s lymphoma cells, and determine site-specific glycosylation changes that occurred upon inhibition of fucosylation at high confidence.

scholarly journals SugarQuant: a streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics

2020 ◽  
Author(s):  
Pan Fang ◽  
Yanlong Ji ◽  
Ivan Silbern ◽  
Carmen Doebele ◽  
Momchil Ninov ◽  
...  
Keyword(s):  
Database Search ◽  
High Confidence ◽  
Site Specific ◽  
Glycosylation Sites ◽  
Data Processing Tool ◽  
A Cell ◽  
One Step ◽  
Global Identification ◽  
Specific Regulation ◽  
Accurate Quantification

AbstractSite-specific regulation of protein N-glycosylation is essential in human cells. However, accurate quantification of glycosylation sites and their individual glycan moieties in a cell-wide manner is still technically challenging. Here, we introduce SugarQuant, an integrated mass spectrometry-based pipeline comprising fast protein aggregation capture (PAC)-based sample preparation, optimized multi-notch MS3 LC-MS acquisition (Glyco-SPS-MS3) and a data-processing tool (GlycoBinder) that allows for confident, global identification and quantification of intact glycopeptides in complex biological samples. PAC greatly reduces the overall samplehandling time without compromising sensitivity. Glyco-SPS-MS3 combines high-resolution MS2 and MS3 scans, resulting in enhanced reporter signals of isobaric mass tags, improved detection of N-glycopeptide fragments, and significantly lowered interference in multiplexed quantification. GlycoBinder enables streamlined processing of Glyco-SPS-MS3 data, followed by a two-step database search which increases the identification rates of intact glycopeptides by up to 22% when compared with one-step database search strategies. SugarQuant was applied to identify and quantify more than 5,000 unique glycoforms in Burkitt’s lymphoma cells, and determined complex site-specific glycosylation changes that occurred upon inhibition of fucosylation at high confidence.

scholarly journals Large-Scale Production of Bioactive Terrein by Aspergillus terreus Strain S020 Isolated from the Saudi Coast of the Red Sea

Biomolecules ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 480 ◽  
Author(s):  
Hani Z. Asfour ◽  
Zuhier A. Awan ◽  
Alaa A. Bagalagel ◽  
Mahmoud A. Elfaky ◽  
Reda F. A. Abdelhameed ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
Crude Extract ◽  
Large Scale ◽  
Aspergillus Terreus ◽  
Antimicrobial Activities ◽  
Carcinoma Cells ◽  
Small Scale ◽  
Scale Production ◽  
Fungal Strains

The diversity of symbiotic fungi derived from two marine sponges and sediment collected off Obhur, Jeddah (Saudi Arabia), was investigated in the current study. A total of 23 isolates were purified using a culture-dependent approach. Using the morphological properties combined with internal transcribed spacer-rDNA (ITS-rDNA) sequences, 23 fungal strains (in the majority Penicillium and Aspergillus) were identified from these samples. The biological screening (cytotoxic and antimicrobial activities) of small-scale cultures of these fungi yielded several target fungal strains which produced bioactive secondary metabolites. Amongst these isolates, the crude extract of Aspergillus terreus strain S020, which was cultured in fermentation static broth, 21 L, for 40 days at room temperature on potato dextrose broth, displayed strong antimicrobial activities against Pseudomonas aeruginosa and Staphylococcus aureus and significant antiproliferative effects on human carcinoma cells. Chromatographic separation of the crude extract by silica gel column chromatography indicated that the S020 isolate could produce a series of chemical compounds. Among these, pure crystalline terrein was separated with a high yield of 537.26 ± 23.42 g/kg extract, which represents the highest fermentation production of terrein to date. Its chemical structure was elucidated on the basis of high-resolution electrospray ionization mass spectrometry (HRESIMS) or high-resolution mass spectrometry (HRMS), 1D, and 2D NMR spectroscopic analyses and by comparison with reported data. The compound showed strong cytotoxic activity against colorectal carcinoma cells (HCT-116) and hepatocellular carcinoma cells (HepG2), with IC50 values of 12.13 and 22.53 µM, respectively. Our study highlights the potential of A. terreus strain S020 for the industrial production of bioactive terrein on a large scale and the importance of future investigations of these strains to identify the bioactive leads in these fungal extracts.

scholarly journals Mass spectrometry–based proteomics in cell biology

2010 ◽  
Vol 190 (4) ◽  
pp. 491-500 ◽  
Author(s):  
Tobias C. Walther ◽  
Matthias Mann
Keyword(s):  
Mass Spectrometry ◽  
Quantitative Analysis ◽  
High Resolution ◽  
Sample Preparation ◽  
Cell Biology ◽  
Computational Analysis ◽  
Global Analysis ◽  
Protein Composition ◽  
Broad Community

The global analysis of protein composition, modifications, and dynamics are important goals in cell biology. Mass spectrometry (MS)–based proteomics has matured into an attractive technology for this purpose. Particularly, high resolution MS methods have been extremely successful for quantitative analysis of cellular and organellar proteomes. Rapid advances in all areas of the proteomic workflow, including sample preparation, MS, and computational analysis, should make the technology more easily available to a broad community and turn it into a staple methodology for cell biologists.

scholarly journals Combining high resolution and exact calibration to boost statistical power: A well-calibrated score function for high-resolution MS2 data

2018 ◽  
Author(s):  
Andy Lin ◽  
J. Jeffry Howbert ◽  
William Stafford Noble
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
Statistical Power ◽  
State Of The Art ◽  
Score Function ◽  
Shotgun Proteomics ◽  
Database Search ◽  
Mass Spectrometry Data ◽  
P Value ◽  
Score Functions

AbstractTo achieve accurate assignment of peptide sequences to observed fragmentation spectra, a shotgun proteomics database search tool must make good use of the very high resolution information produced by state-of-the-art mass spectrometers. However, making use of this information while also ensuring that the search engine’s scores are well calibrated—i.e., that the score assigned to one spectrum can be meaningfully compared to the score assigned to a different spectrum—has proven to be challenging. Here, we describe a database search score function, the “residue evidence” (res-ev) score, that achieves both of these goals simultaneously. We also demonstrate how to combine calibrated res-ev scores with calibrated XCorr scores to produce a “combined p-value” score function. We provide a benchmark consisting of four mass spectrometry data sets, which we use to compare the combined p-value to the score functions used by several existing search engines. Our results suggest that the combined p-value achieves state-of-the-art performance, generally outperforming MS Amanda and Morpheus and performing comparably to MS-GF+. The res-ev and combined p-value score functions are freely available as part of the Tide search engine in the Crux mass spectrometry toolkit (http://crux.ms).

scholarly journals Large-scale, enzyme-based xenobiotic identification for exposomics

2020 ◽  
Author(s):  
Ken Liu ◽  
Choon Lee ◽  
Grant Singer ◽  
Michael Woodworth ◽  
Thomas Ziegler ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
Retention Time ◽  
Human Disease ◽  
Large Scale ◽  
High Resolution Mass Spectrometry ◽  
Chemical Identification ◽  
Mass Spectrometry Identification ◽  
Level Of Understanding ◽  
Resolution Mass

Abstract Advances in genomics have revealed many of the genetic underpinnings of human disease, but exposomics methods are currently inadequate to obtain a similar level of understanding of environmental contributions to human disease. Exposomics methods are limited by low abundance of xenobiotic metabolites and lack of authentic standards, which precludes identification using solely mass spectrometry-based criteria. Here, we develop and validate a method for enzymatic generation of xenobiotic metabolites for use with high-resolution mass spectrometry (HRMS) for chemical identification. Generated xenobiotic metabolites were used to confirm identities of respective metabolites in mice and human samples based upon accurate mass, retention time and co-occurrence with related xenobiotic metabolites. The results establish a generally applicable enzyme-based identification (EBI) for mass spectrometry identification of xenobiotic metabolites.

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