Electron Transfer Dissociation for Biopolymer Sequence Analysis

Abstract

The present invention relates to a new method for fragmenting ions in a mass spectrometer through the use of electron transfer dissociation, and for performing sequence analysis of peptides and proteins by mass spectrometry. In the case of peptides, the invention promotes fragmentation along the peptide backbone and makes it possible to deduce the amino acid sequence of the sample, including modified amino acid residues, through the use of an RF field device.

1 . A method for dissociating multiply charged organic and/or biomolecular cations for mass spectrometry analysis, said method comprising the steps of
 introducing said multiply charged cations into an RF electric field ion containment device;   introducing anions as gas-phase electron transfer reagents into said ion containment device;   mixing the introduced anions, or derivative anions thereof, and the multiply charged cations, or derivative multiply charged cations thereof, wherein said derivative anions and said derivative multiply charged cations are generated within said ion containment device during performance of said method, so as to facilitate electron transfer from the anions, or said derivative anions thereof, to the multiply charged cations, or derivative multiply charged cations thereof, to induce cleavage of covalent bonds and produce fragment and/or dissociation product cations.   
     
     
         2 . The method of  claim 1  wherein the multiply charged cation is a polypeptide. 
     
     
         3 . The method of  claim 1  wherein the RF electric field ion containment device is an RF ion guide. 
     
     
         4 . The method of  claim 1  wherein the RF electric field ion containment device is an RF ion trap. 
     
     
         5 . The method of  claim 4  wherein the RF ion trap is a RF linear multipole ion trap. 
     
     
         6 . The method of  claim 4  wherein the RF ion trap is a RF 3 dimensional multipole ion trap. 
     
     
         7 . The method of  claim 1  wherein the kinetic energies of the introduced anions, or said derivative anions thereof, and the multiply charged cations, or said derivative multiply charged cations thereof, are less than 1 electron volt, during the mixing step. 
     
     
         8 . The method of  claim 1  wherein said anions and/or said derivative anions are radical anions. 
     
     
         9 . The method of  claim 5  where said anions are injected along the linear axis of the RF linear multipole ion trap. 
     
     
         10 . The method of  claim 1  wherein the gas-phase anions are generated from a polyaromatic hydrocarbon and said method further comprises the step of
 mass (m/z) analyzing and detecting said fragment and/or dissociation product cations or cations derived from said fragment and/or dissociation product cations for mass spectrometric analysis.   
     
     
         11 . The method of  claim 10  wherein the polyaromatic hydrocarbon anions are generated from a low electron affinity substrate selected from the group consisting of anthracene, 9,10-diphenyl-anthracene, naphthalene, fluorene, phenanthrene, pyrene, fluoranthene, chrysene, triphenylene, perylene, acridine, 2,2′-dipyridyl, 2,2′-biquinoline, 9-anthracenecarbonitrile, dibenzothiophene, 1,10′-phenanthroline, 9′-anthracenecarbonitrile, and anthraquinone and substituted derivatives of said low electron affinity substrates. 
     
     
         12 . The method of  claim 5  wherein the ion trap is a segmented linear RF multipole ion trap. 
     
     
         13 . The method of  claim 5  wherein the gas-phase reagent anions are radical gas-phase anions generated from polyaromatic hydrocarbon or substituted polyaromatic hydrocarbon compounds. 
     
     
         14 . The method of  claim 10  wherein the multiply charged cations and the polyaromatic hydrocarbon anions are spatially segregated within an RF field ion containment device until said mixing step. 
     
     
         15 . The method of  claim 10  wherein the multiply charged polypeptide cations and the polyaromatic hydrocarbon anions are spatially segregated within an ion trap until said mixing step. 
     
     
         16 . The method of  claim 2  further comprising an additional activation step wherein sufficient energy in the form of photoactivation or collisional activation is supplied to the undissociated charge reduced electron transfer product cations so as to promote dissociation of said undissociated charge reduced electron transfer product cations via electron transfer-type dissociation pathway, with less than a 20% production of fragment cations characteristic of collisional-activation. 
     
     
         17 . The method of  claim 2  further comprising an additional activation step wherein sufficient energy in the form of photoactivation or collisional activation is supplied to the undissociated charge reduced electron transfer product cations so as to promote dissociation of said undissociated charge reduced electron transfer product cations via electron transfer-type dissociation pathway, with less than a 5% production of fragment cations characteristic of collisional-activation. 
     
     
         18 . A method for dissociating a polypeptide by negative electron transfer dissociation (NETD) for mass spectrometry analysis, said method comprising
 introducing multiply charged polypeptide anions into an RF electric field ion containment device;   introducing gas-phase cations, into an RF electric field ion containment device;   mixing the gas-phase cations and the polypeptide anions so as to facilitate electron transfer from the anions to the cations, and thus inducing the cleavage of covalent bonds and production of negative electron transfer fragment and/or dissociation product anions; and   mass (m/z) analyzing and detecting said negative electron transfer dissociation product ions or ions derived from negative electron transfer dissociation product ions.   
     
     
         19 . The method of  claim 18  wherein the cations are selected from the group consisting of inert gas cations. 
     
     
         20 . The method of  claim 10  wherein said polyaromatic hydrocarbon anions and/or said derivative anions are radical anions.