Rapid protein sequencing by the enzyme-thermospray liquid chromatographic/mass spectrometric method

K, Stachowiak; Wilder, Cheryl; Vestal, Marvin L.; Dyckes, Douglas F.

doi:10.1021/ja00214a017

Cited by 36 publications

(12 citation statements)

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“…continuous-flow fast-atom bombardment and ion spray) have detected these and other similar synthetic peptides on the order of picomoles as well (24,25). Our results are comparable to these other techniques, and they represent an enhancement of 1 to 2 orders of magnitude in sensitivity when compared to a thermospray ion source without a repeller and a restricted probe tip (9,10,26).…”

Section: Resultssupporting

confidence: 73%

“…Previous models of the thermospray ion source (without a needle-tip repeller and a restricted probe tip) required from 0.2 to 10 nmol of peptides for detection. The amount of peptide sample needed for previous on-line column thermospray studies was typically on the order of 1-10 nmol (9,10). The amount of sample required to produce spectra by direct injection (without an on-line column) was typically 200-500 pmol or more (9,10).…”

Section: Resultsmentioning

confidence: 99%

“…The amount of peptide sample needed for previous on-line column thermospray studies was typically on the order of 1-10 nmol (9,10). The amount of sample required to produce spectra by direct injection (without an on-line column) was typically 200-500 pmol or more (9,10).…”

Section: Resultsmentioning

confidence: 99%

“…Although ion evaporation predominates for compounds that are multiply charged in solution, other compounds have been shown to undergo ionization by gas-phase ion/molecule reactions in the thermospray ion source (7)(8)(9)(10). Dual-beam thermospray experiments have shown that a number of compounds are ionized via chemical ionization (Cl) (7).…”

Section: Introductionmentioning

confidence: 99%

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Enhanced analysis of poly(ethylene glycols) and peptides using thermospray mass spectrometry

Fink¹,

Freas²

1989

Anal. Chem.

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The effects of a needle-tip repeller electrode on thermospray mass spectra of poly(ethylene glycols) and peptide samples have been studied. A significant increase in ion current was observed for increased repeller potentials in the low-mass region (m/z 300 to m/z 1100) and the high-mass region (m/z 1100 to m/z 1900). The change in ion current is due to a change in ion extraction efficiency because of increased ion diffusion rates. The amount of peptide sample needed for direct injection studies was decreased 1 to 2 orders of magnitude by using an ion source with both a needle-tip repeller electrode and a restricted vaporizer probe tip.

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Section: Resultssupporting

confidence: 73%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Enhanced analysis of poly(ethylene glycols) and peptides using thermospray mass spectrometry

Fink¹,

Freas²

1989

Anal. Chem.

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“…Efforts have been made in the past to investigate the effectiveness of coupling a microreactor containing immobilized proteolytic enzyme to analytical tools such as LC and/or mass spectrometry. 15,17,19,21,24,30 The approach was found to be advantageous in a number of ways including shorter digestion time, higher cleavage efficiency and less autolysis. Therefore, we have developed a method where the digestion is performed by linking a cartridge packed with immobilized pepsin media on-line with LC/ MS. Digestion was accomplished when the enzyme was infused through the cartridge.…”

Section: On-line Pepsin Digestionmentioning

confidence: 99%

Identification of the protein-drug adduct formed between aldehyde dehydrogenase andS-methyl-N,N-diethylthiocarbamoyl sulfoxide by on-line proteolytic digestion high performance liquid chromatography electrospray ionization mass spectrometry

Shen

Johnson

Mays

et al. 2000

Rapid Commun. Mass Spectrom.

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Disulfiram has been used clinically as an aversion therapy treatment for recovering alcoholics. One of its metabolites, S-methyl-N, N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), is currently believed by some to be the active metabolite in vivo. We demonstrate in this report that MeDTC-SO is a potent irreversible inhibitor of recombinant rat liver mitochondrial aldehyde dehydrogenase (rlmALDH), the enzyme responsible for oxidizing acetaldehyde formed during ethanol metabolism. Recombinant rlmALDH was inhibited by MeDTC-SO after in vitro incubation with an IC(50) = 4.62 microM. The inhibition of rlmALDH was found to be accompanied by a concomitant increase of approximately 100 Da to the molecular mass of the native enzyme as determined by on-line high performance liquid chromatography (HPLC) electrospray ionization mass spectrometry (LC/MS), indicating that a covalent modification has occurred. To determine the site and structure of this covalent adduct, we developed a novel approach to characterize specific protein-drug interactions by linking a proteolytic enzyme digestion cartridge on-line with LC/MS. The on-line pepsin digestion LC/MS of MeDTC-SO-inhibited rlmALDH revealed an ion at MH(2)(2+) = 500.9, which was not present in the pepsin digestion of the non-inhibited enzyme. This peptide was tentatively attributed to the putative active site peptide (FNQGQC(301)C(302)C(303)) plus the adduct. This peptide was subjected to analysis by LC/MS/MS, which allowed us to determine that the covalent modification was associated with a single carbamoyl adduct at Cys-302, which has been shown to be the active site nucleophile of the enzyme.

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Peptide Sequencing by Mass Spectrometry

Stults¹

1990

Methods of Biochemical Analysis

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Rapid protein sequencing by the enzyme-thermospray liquid chromatographic/mass spectrometric method

Cited by 36 publications

References 0 publications

Enhanced analysis of poly(ethylene glycols) and peptides using thermospray mass spectrometry

Enhanced analysis of poly(ethylene glycols) and peptides using thermospray mass spectrometry

Identification of the protein-drug adduct formed between aldehyde dehydrogenase andS-methyl-N,N-diethylthiocarbamoyl sulfoxide by on-line proteolytic digestion high performance liquid chromatography electrospray ionization mass spectrometry

Peptide Sequencing by Mass Spectrometry

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