Mass Spectral Detection of Diethoxyphospho-Tyrosine Adducts on Proteins from HEK293 Cells Using Monoclonal Antibody depY for Enrichment

Onder, Seda; Schopfer, Lawrence M.; Tacal, Özden; Blake, Thomas A.; Johnson, Rudolph C.; Lockridge, Oksana

doi:10.1021/acs.chemrestox.8b00083

Cited by 15 publications

(16 citation statements)

References 38 publications

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“…A 5 μL aliquot of the digest representing approximately 5 μg of protein was analyzed on the 6600 Triple-TOF mass spectrometer using a data-directed fragmentation method, as described [16]. Data were searched against the UniProt protein sequence database (species Homo sapiens) using the Paragon algorithm v4.5 from Protein Pilot software v4.0 to match observed peptides to sequences in the database.…”

Section: Methodsmentioning

confidence: 99%

Purification of human butyrylcholinesterase from frozen Cohn fraction IV-4 by ion exchange and Hupresin affinity chromatography

et al. 2019

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Human butyrylcholinesterase (HuBChE) is being developed as a therapeutic for protection from the toxicity of nerve agents. An enriched source of HuBChE is Cohn fraction IV-4 from pooled human plasma. For the past 40 years, purification of HuBChE has included affinity chromatography on procainamide-Sepharose. The present report supports a new affinity sorbent, Hupresin, for purification of HuBChE from Cohn fraction IV-4. Nine batches of 70–80 kg frozen Cohn fraction were extracted with water, filtered, and chromatographed on 30 L of Q-Ceramic ion exchange sorbent at pH 4.5. The 4% pure Q-eluent was pumped onto 4.2 L Hupresin, where contaminants were washed off with 0.3 M NaCl in 20 mM sodium phosphate pH 8.0, before 99% pure HuBChE was eluted with 0.1 M tetramethylammonium bromide. The average yield was 1.5 g of HuBChE from 80 kg Cohn paste. Recovery of HuBChE was reduced by 90% when the paste was stored at -20°C for 1 year, and reduced 100% when stored at 4°C for 24h. No reduction in HuBChE recovery occurred when paste was stored at -80°C for 3 months or 3 years. Hupresin and procainamide-Sepharose were equally effective at purifying HuBChE from Cohn fraction. HuBChE in Cohn fraction required 1000-fold purification to attain 99% purity, but 15,000-fold purification when the starting material was plasma. HuBChE (P06276) purified from Cohn fraction was a 340 kDa tetramer of 4 identical N-glycated subunits, stable for years in solution or as a lyophilized product.

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Section: Methodsmentioning

confidence: 99%

Purification of human butyrylcholinesterase from frozen Cohn fraction IV-4 by ion exchange and Hupresin affinity chromatography

et al. 2019

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“…The protocol for liquid chromatography tandem mass spectrometry (LC–MS/MS) is described in detail in [11]. In brief, peptides in a 5 µL volume were separated on a cHiPLC Nanoflex microchip column (Eksigent, Dublin, CA) packed with ChromXP C18.…”

Section: Experimental Design Materials and Methodsmentioning

confidence: 99%

Tetramer organizing polyproline-rich peptides identified by mass spectrometry after release of the peptides from Hupresin-purified butyrylcholinesterase tetramers isolated from milk of domestic pig (Sus scrofa)

et al. 2018

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Milk of the domestic pig has 10 times more butyrylcholinesterase (BChE) per mL than porcine serum. We purified BChE from porcine milk by affinity chromatography on Hupresin-Sepharose. The pure porcine BChE (PoBChE) was a tetramer with a molecular weight of 340,000, similar to that of human BChE tetramers. The C-terminal 40 residues of PoBChE constitute the tetramerization domain. The glue that holds the 4 BChE subunits together is a polyproline-rich peptide. Mass spectrometry analysis of trypsin-digested PoBChE identified a variety of polyproline-rich peptides originating from 12 different proteins. The donor proteins exist in the nucleus or cytoplasm of cells and contribute their polyproline-rich peptides after a cell is degraded. The secreted PoBChE scavenges the polyproline-rich peptides and incorporates one polyproline peptide per PoBChE tetramer, where the polyproline peptide is bound noncovalently but very tightly with an estimated dissociation constant of 10–12 M. The most abundant polyproline-rich peptides were derived from acrosin, homeobox protein HoxB4, lysine-specific demethylase 6B, proline-rich protein 12, and proline-rich membrane anchor 1 (PRiMA). The research article associated with the data in this report can be found in Saxena et al. (2018). The Data in Brief report lists all the polyproline-rich peptides identified in PoBChE tetramers.

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“…A limited number of reports had appeared describing reactions of OP with tyrosine in proteins such as human serum albumin (15), bromelain (16), papain (17), and hen egg white lysozyme (18). Beginning in 2007 we confirmed and amplified these observations using MS. We found OP adducts on both tyrosine and lysine in tubulin, albumin, casein, aprotinin, and keratin (19–23). The OP in these studies included soman, sarin, chlorpyrifos oxon, dichlorvos, diisopropylfluorophosphate, and a biotinylated OP probe, FP-biotin.…”

Section: Introductionmentioning

confidence: 92%

Chlorpyrifos oxon promotes tubulin aggregation via isopeptide cross-linking between diethoxyphospho-Lys and Glu or Asp: Implications for neurotoxicity

Schopfer

Lockridge

2018

Journal of Biological Chemistry

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Exposure to organophosphorus toxicants (OP) can have chronic adverse effects that are not explained by inhibition of acetylcholinesterase, the cause of acute OP toxicity. We therefore hypothesized that OP-induced chronic illness is initiated by the formation of organophosphorus adducts on lysine residues in proteins, followed by protein cross-linking and aggregation. Here, Western blots revealed that exposure to the OP chlorpyrifos oxon converted porcine tubulin from its original 55-kDa mass to high-molecular-weight aggregates. Liquid chromatography–tandem MS analysis of trypsin-digested samples identified several diethoxyphospho-lysine residues in the OP-treated tubulin. Using a search approach based on the Batch Tag program, we identified cross-linked peptides and found that these chemically activated lysines reacted with acidic amino acid residues creating γ-glutamyl-ϵ-lysine or aspartyl-ϵ-lysine isopeptide bonds between β- and α-tubulin. Of note, these cross-linked tubulin molecules accounted for the high-molecular-weight aggregates. To the best of our knowledge, this is the first report indicating that chlorpyrifos oxon–exposed tubulin protein forms intermolecular cross-links with other tubulin molecules, resulting in high-molecular-weight protein aggregates. It is tempting to speculate that chronic illness from OP exposure may be explained by a mechanism that starts with OP adduct formation on protein lysines followed by protein cross-linking. We further speculate that OP-modified or cross-linked tubulin can impair axonal transport, reduce neuron connections, and result in neurotoxicity.

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Mass Spectral Detection of Diethoxyphospho-Tyrosine Adducts on Proteins from HEK293 Cells Using Monoclonal Antibody depY for Enrichment

Cited by 15 publications

References 38 publications

Purification of human butyrylcholinesterase from frozen Cohn fraction IV-4 by ion exchange and Hupresin affinity chromatography

Purification of human butyrylcholinesterase from frozen Cohn fraction IV-4 by ion exchange and Hupresin affinity chromatography

Tetramer organizing polyproline-rich peptides identified by mass spectrometry after release of the peptides from Hupresin-purified butyrylcholinesterase tetramers isolated from milk of domestic pig (Sus scrofa)

Chlorpyrifos oxon promotes tubulin aggregation via isopeptide cross-linking between diethoxyphospho-Lys and Glu or Asp: Implications for neurotoxicity

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