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Simultaneous sequence analysis of amino- and carboxy-termini

A carboxyl-terminal, amino-terminal technique, applied in the field of simultaneous sequence analysis of amino and carboxyl-terminal ends

Inactive Publication Date: 2007-11-14
UNIV OF VIRGINIA ALUMNI PATENTS FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the randomness and unpredictability of CAD breaks, this experiment has not yet become a routine tool for whole protein sequence identification

Method used

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  • Simultaneous sequence analysis of amino- and carboxy-termini
  • Simultaneous sequence analysis of amino- and carboxy-termini
  • Simultaneous sequence analysis of amino- and carboxy-termini

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Experimental program
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Embodiment approach

[0041] According to the present disclosure, there is provided a method for rapid sequence analysis of the amino and carboxyl termini of intact proteins or large protein degradation products using mass spectrometry and multiple ion / ion reactions. In one embodiment, the method provides a rapid means of identifying or confirming the presence of a polypeptide 30, 40, 50 or more amino acids in length from a sample. A sample may contain the polypeptide of interest in pure form (eg, greater than 99% pure), or may contain other polypeptides or compounds. Samples can also be derived from complex mixtures of other proteins / peptides, such as whole cell lysates. More particularly, in one embodiment, the method comprises the steps of performing a first ion-ion reaction (electron transfer dissociation: ETD) to dissociate polypeptide ions in a mass spectrometry system, and then performing a second A secondary ion-ion reaction (proton transfer reaction: PTR) to reduce the charge of the ion f...

Embodiment 1

[0168] Use of negative ions for electron transfer dissociation of polypeptides

[0169] According to one embodiment, FC-43 (perfluorotributylamine, PFTBA), sulfur hexafluoride (SF 6 ), perfluoro-1,3-dimethylcyclohexane (PDCH), benzene hexafluoride (C 6 f 6 ) and anthracene were introduced into a NICI (Negative Ion Chemical Ionization) source to generate negative ions for the experiment. In all cases, the negative ions generated in the aforementioned sources produced at least some ETD products when reacted with standard peptide precursor ions. When the standard m / z calibrant FC-43 for electron impact ionization source mass spectrometers was introduced into the source, only small amounts of c and z ions were generated with very low conversion efficiency of precursors to ETD products. In later experiments, the above molecules were introduced individually into the ion source and both produced extensive c- and z-type fragmentation of our standard precursor ion, a triple-charged ...

Embodiment 2

[0174] Continuous Ion / Ion Reaction

[0175] As mentioned above, a certain negative ion mainly acts as ETD or PTR reactant. The individual reactions described above can be performed separately and sequentially by exposing positive ions to negative ions of either species. For example, highly charged peptide precursor ions (eg, z>4) can be dissociated with an ETD-inducing anion, followed by removal of those reactants and introduction of a second PTR-inducing anion. The duration of the second reaction can be adjusted so that the charge state of the product species is reduced in a controlled manner. That is, a +10 precursor peptide can be dissociated by ETD to produce fragments charged from +1 to +9. Of course, m / z resolution of the isotopic peaks of such highly charged products can be problematic; so the duration of the secondary PTR reaction can be adjusted so that the ETD product converts to a predominantly +1 charged state. The net effect of the above reactions is to convert...

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Abstract

The present invention relates to a new method for identifying polypeptides by deducing the amino acid sequence of the carboxy and amino termini by a mass spectrometer analysis. The method comprises the steps of dissociating highly charged peptide precursor ions (e.g., z > 4) using electron transfer dissociation inducing anions followed by removal of those reagents and introduction of a second, proton transfer inducing anion type. The second PTR reaction duration is adjusted to convert the ETD products to primarily the + 1 charge-state to reduce the highly charged c and z-type fragments, producing an m / z spectrum containing a series of c and z-type fragment ions that are easily interpreted to reveal the sequence of the amino and carboxy terminus, respectively.

Description

[0001] related application [0002] This application claims priority under 35 USC § 119(e) from US Provisional Application Serial No. 60 / 617,125 filed October 8, 2004, the disclosures of which are incorporated herein by reference. Background technique [0003] The identification and characterization of proteins and peptides has become an important branch of modern biology, and mass spectrometry has become one of the most important means for analyzing peptides and proteins. A new method for the dissociation of peptide ions known as electron transfer dissociation (ETD) has recently been reported (Syka, J.E.P.; Coon, J.J.; Schroeder, M.J.; Shabanowitz, J.; Hunt, D.F. Proceedings of the National Academy of Sciences of the United States of America 2004, 101, 9528-9533; Coon, J.J.; Syka, J.E.P.; Schwartz, J.C; Shabanowitz, J.; Hunt, D.F. International Journal of Mass Spectrometry 2004, in press 2004). In ETD, negative ions react with multiply protonated peptide / protein positive ion...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/6848Y10T436/25875Y10T436/24G01N33/6818H01J49/02
Inventor D·F·亨特J·J·库恩J·E·P·塞克
Owner UNIV OF VIRGINIA ALUMNI PATENTS FOUND
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