scholarly journals Top-Down Proteomic Identification of Shiga Toxin 1 and 2 from Pathogenic Escherichia coli Using MALDI-TOF-TOF Tandem Mass Spectrometry

2019 ◽  
Vol 7 (11) ◽  
pp. 488 ◽  
Author(s):  
Clifton K. Fagerquist ◽  
William J. Zaragoza ◽  
Michelle Q. Carter
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Tandem Mass Spectrometry ◽  
Disulfide Bond ◽  
Shiga Toxin ◽  
Tandem Mass ◽  
Top Down ◽  
E Coli ◽  
B Subunit ◽  
Proteomic Identification

Shiga-toxin-producing Escherichia coli (STEC) are a burden on agriculture and a threat to public health. Rapid methods are needed to identify STEC strains and characterize the Shiga toxin (Stx) they produce. We analyzed three STEC strains for Stx expression, using antibiotic induction, matrix-assisted laser desorption/ionization time-of-flight-time-of-flight (MALDI-TOF-TOF) mass spectrometry, and top-down proteomic analysis. E. coli O157:H- strain 493/89 is a clinical isolate linked to an outbreak of hemolytic uremic syndrome (HUS) in Germany in the late 1980s. E. coli O145:H28 strains RM12367-C1 and RM14496-C1 were isolated from an agricultural region in California. The stx operon of the two environmental strains were determined by whole genome sequencing (WGS). STEC strain 493/89 expressed Shiga toxin 2a (Stx2a) as identified by tandem mass spectrometry (MS/MS) of its B-subunit that allowed identification of the type and subtype of the toxin. RM12367-C1 also expressed Stx2a as identified by its B-subunit. RM14496-C1 expressed Shiga toxin 1a (Stx1a) as identified from its B-subunit. The B-subunits of Stx1 and Stx2 both have an intramolecular disulfide bond. MS/MS was obtained on both the disulfide-bond-intact and disulfide-bond-reduced B-subunit, with the latter being used for top-down proteomic identification. Top-down proteomic analysis was consistent with WGS.

scholarly journals Top-down proteomic identification of plasmid and host proteins produced by pathogenic Escherichia coli using MALDI-TOF-TOF tandem mass spectrometry

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0260650
Author(s):  
Clifton K. Fagerquist ◽  
Claire E. Dodd
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Tandem Mass Spectrometry ◽  
Aspartic Acid ◽  
Protein Sequences ◽  
Tandem Mass ◽  
Host Proteins ◽  
Top Down ◽  
Maldi Tof ◽  
Fragment Ions

Fourteen proteins produced by three pathogenic Escherichia coli strains were identified using antibiotic induction, MALDI-TOF-TOF tandem mass spectrometry (MS/MS) and top-down proteomic analysis using software developed in-house. Host proteins as well as plasmid proteins were identified. Mature, intact protein ions were fragmented by post-source decay (PSD), and prominent fragment ions resulted from the aspartic acid effect fragmentation mechanism wherein polypeptide backbone cleavage (PBC) occurs on the C-terminal side of aspartic acid (D), glutamic acid (E) and asparagine (N) residues. These highly specific MS/MS-PSD fragment ions were compared to b- and y-type fragment ions on the C-terminal side of D-, E- and N-residues of in silico protein sequences derived from whole genome sequencing. Nine proteins were found to be post-translationally modified with either removal of an N-terminal methionine or a signal peptide. The protein sequence truncation algorithm of our software correctly identified all full and truncated protein sequences. Truncated sequences were compared to those predicted by SignalP. Nearly complete concurrence was obtained except for one protein where SignalP mis-identified the cleavage site by one residue. Two proteins had intramolecular disulfide bonds that were inferred by the absence of PBC on the C-terminal side of a D-residue located within the disulfide loop. These results demonstrate the utility of MALDI-TOF-TOF for identification of full and truncated bacterial proteins.

scholarly journals Characterization of a novel + 70 Da modification in rhGM-CSF expressed in E. coli using chemical assays in combination with mass spectrometry

Amino Acids ◽  
2021 ◽  
Author(s):  
Magdalena Widgren Sandberg ◽  
Jakob Bunkenborg ◽  
Stine Thyssen ◽  
Martin Villadsen ◽  
Thomas Kofoed
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Peptide Mapping ◽  
Covalent Modification ◽  
Peptide Fragmentation ◽  
Tandem Mass ◽  
E Coli ◽  
Gm Csf ◽  
Fragmentation Behavior ◽  
Macrophage Colony

AbstractGranulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine and a white blood cell growth factor that has found usage as a therapeutic protein. During analysis of different fermentation batches of GM-CSF recombinantly expressed in E. coli, a covalent modification was identified on the protein by intact mass spectrometry. The modification gave a mass shift of + 70 Da and peptide mapping analysis demonstrated that it located to the protein N-terminus and lysine side chains. The chemical composition of C4H6O was found to be the best candidate by peptide fragmentation using tandem mass spectrometry. The modification likely contains a carbonyl group, since the mass of the modification increased by 2 Da by reduction with borane pyridine complex and it reacted with 2,4-dinitrophenylhydrazine. On the basis of chemical and tandem mass spectrometry fragmentation behavior, the modification could be attributed to crotonaldehyde, a reactive compound formed during lipid peroxidation. A low recorded oxygen pressure in the reactor during protein expression could be linked to the formation of this compound. This study shows the importance of maintaining full control over all reaction parameters during recombinant protein production.

scholarly journals Nano-LC FTICR Tandem Mass Spectrometry for Top-Down Proteomics: Routine Baseline Unit Mass Resolution of Whole Cell Lysate Proteins up to 72 kDa

2012 ◽  
Vol 84 (5) ◽  
pp. 2111-2117 ◽  
Author(s):  
Jeremiah D. Tipton ◽  
John C. Tran ◽  
Adam D. Catherman ◽  
Dorothy R. Ahlf ◽  
Kenneth R. Durbin ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Mass Resolution ◽  
Unit Mass ◽  
Tandem Mass ◽  
Top Down ◽  
Whole Cell ◽  
Whole Cell Lysate ◽  
Cell Lysate ◽  
Unit Mass Resolution

scholarly journals A robust two-dimensional separation for top-down tandem mass spectrometry of the low-mass proteome

2009 ◽  
Vol 20 (12) ◽  
pp. 2183-2191 ◽  
Author(s):  
Ji Eun Lee ◽  
John F. Kellie ◽  
John C. Tran ◽  
Jeremiah D. Tipton ◽  
Adam D. Catherman ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Tandem Mass ◽  
Two Dimensional ◽  
Top Down ◽  
Low Mass
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