Methods for sequencing biopolymers

Abstract

This invention is directed to methods for sequencing biopolymers. Mass-defined biopolymers are immobilized on a probe and detected by mass spectrometry.

What is claimed:  
     
         72 . A method for biopolymer sequence determination comprising the steps of: 
 binding a biopolymer analyte to probe tip containing a sample presenting surface having a surface selected molecule selected from the group consisting of an energy absorbing molecule, an affinity capture device, a photolabile attachment molecule and a combination thereof;    desorption of biopolymer analyte in mass spectrometry analysis, wherein at least a portion of said biopolymer is not desorbed from the probe tip;    analyzing the results of the desorption modifying the biopolymer analyte still bound to the probe tip; and    repeating the desorption, analyzing and modifying steps until the biopolymer is sequenced.    
     
     
         73 . The method of  claim 72 , wherein the biopolymer is selected from the group consisting of protein, RNA, DNA and carbohydrate.  
     
     
         74 . A method for determining the sequence of monomers in a biopolymer selected from nucleic acids, polypeptides and polysaccharides comprising the steps of: 
 a) providing a system comprising: 
 (1) a removably insertable probe having a surface for presenting the analyte to an energy source that emits energy capable of desorbing the analyte from the probe, and an immobilized affinity reagent on the probe surface bound to the analyte, wherein the analyte comprises a population of mass-defined biopolymers modified by having different numbers of monomers cleaved from an end of the biopolymer;  
 (2) an energy source that directs energy to the probe surface for desorbing the analyte; and  
 (3) a detector in communication with the probe surface that detects the desorbed analyte;  
   b) desorbing at least a portion of the analyte from the probe surface by exposing the analyte to energy from the energy source;    c) detecting the desorbed analyte with the detector;    d) determining the difference in mass between detected biopolymers of the population; and    e) determining the sequence of monomers based on the difference in mass between desorbed biopolymers of the population.    
     
     
         75 . The method of  claim 74  wherein the system is a laser desorption mass spectrometer wherein: 
 the energy source emits laser light that causes photolytic cleavage and ionizes the analyte to produce an ion,  
 the device further comprises means for accelerating the ion to the detector,  
 the detector detects the ion, and  
 the device further comprises means for determining the mass of the ion.  
 
     
     
         76 . The method of  claim 74  comprising the step of binding the biopolymer to the immobilized affinity reagent analyte and cleaving monomer units from an end of the biopolymer to produce the population of mass-defined biopolymers.  
     
     
         77 . The method of  claim 74  wherein the affinity reagent is immobilized by binding to the probe surface.  
     
     
         78 . The method of  claim 74  wherein the affinity reagent is immobilized by binding to a solid phase and the method comprises the steps of: 
 exposing the affinity reagent bound to the solid phase to the analyte hereby the analyte binds to the affinity reagent;  
 transferring the solid phase to the probe surface; and  
 removably inserting the probe into the system.  
 
     
     
         79 . The method of  claim 74  wherein the biopolymer is a nucleic acid.  
     
     
         80 . The method of  claim 74  wherein the biopolymer is a protein.  
     
     
         81 . The method of  claim 74  wherein the biopolymer is a polysaccharide.  
     
     
         82 . The method of  claim 74  wherein the probe comprises glass.  
     
     
         83 . The method of  claim 74  wherein the probe comprises ceramic.  
     
     
         84 . The method of  claim 74  wherein the probe comprises a synthetic polymer.  
     
     
         85 . The method of  claim 76  comprising preparing the population of biopolymers by chemical or enzymatic means to produce distinct analyte fragments of the biopolymer.  
     
     
         86 . The method of  claim 76  comprising preparing the population of biopolymers by cleaving monomers from the polymers chemically.  
     
     
         87 . The method of  claim 76  comprising preparing the population of biopolymers by cleaving monomers from the polymers enzymatically.  
     
     
         88 . The method of  claim 78  wherein the solid phase comprises a porous or non-porous bead of cross-linked polymer.  
     
     
         89 . The method of  claim 78  wherein the solid phase comprises a magnetic or paramagnetic material.  
     
     
         90 . The method of  claim 78  wherein the solid phase comprises a polymeric bead to which the affinity reagent is attached.  
     
     
         91 . The method of  claim 79  wherein the nucleic acids are modified by digestion with an exonuclease.  
     
     
         92 . The method of  claim 79  comprising cleavage of nucleic acids with endonuclease.  
     
     
         93 . The method of  claim 80  wherein the polypeptides are modified by exopeptidase digestion or N-terminal degradation by Edman degradation.  
     
     
         94 . The method of  claim 80  comprising cleavage of polypeptides with endoprotease or cyanogen bromide.  
     
     
         95 . The method of  claim 80  wherein the polypeptide is bound to a plurality of different photolabile attachment molecules and the method comprises determining the sequence of monomers in a plurality of fragments of the polypeptide, wherein the fragments are generated by endopeptidase digestion of the polypeptide.  
     
     
         96 . The method of  claim 81  wherein the polysaccharides are modified by digestion with neuraminidase, mannase, fucase, galactosidase, glucosidase, O-glycanase or N-glycanase.  
     
     
         97 . The method of  claim 81  comprising cleavage of the polysaccharides with endoglycosidase H or endoglycosidase F.  
     
     
         98 . The method of  claim 81  wherein the polysaccharides are modified by digestion with FUCase I, MANase I, HEXase I, MANase III or PNGase.  
     
     
         99 . The method of  claim 88  wherein the cross-linked polymer is agarose, cellulose or dextran.  
     
     
         100 . A method for determining the sequence of monomers in a biopolymer selected from nucleic acids, polypeptides and polysaccharides comprising the steps of: 
 a) providing a system comprising: 
 (1) a probe that is removably insertable into a mass spectrometer having a surface for presenting an analyte to an energy source that emits energy capable of desorbing the analyte from the probe for analyte detection, and having an immobilized photolabile attachment molecule on the probe surface, the photolabile attachment molecule comprising a binding site to which the analyte is bound, wherein the analyte is releasable from the probe by photolytic cleavage, wherein the analyte comprises a population of mass-defined biopolymers modified by having different numbers of monomers cleaved from an end of the biopolymer;  
 (2) a light source that directs light to the probe surface causing the photolytic cleavage;  
 (3) an energy source that directs light to the probe surface, the energy desorbing the analyte from the probe surface; and  
 (4) a detector in communication with the probe surface that detects the desorbed analyte;  
   b) desorbing at least a portion of the biopolymers of the population from the probe by (1) exposing the analyte to light from the light source, thereby releasing the analyte from the probe by photolytic cleavage; and (2) exposing the analyte to energy from the energy source,    (c) detecting desorbed biopolymers with the detector;    (d) determining the difference in mass between detected biopolymers of the population; and    (e) determining the sequence of monomers based on the difference in mass between desorbed biopolymers of the population.    
     
     
         101 . The method of  claim 100  wherein the system is a laser desorption mass spectrometer wherein: 
 the energy source emits laser light that causes photolytic cleavage and ionizes the analyte to produce an ion,  
 the device further comprises means for accelerating the ion to the detector,  
 the detector detects the ion, and  
 the device further comprises means for determining the mass of the ion.  
 
     
     
         102 . The method of  claim 100  wherein the photolabile attachment molecules are covalently bound to the probe surface.  
     
     
         103 . The method of  claim 100  wherein the photolabile attachment molecules are covalently bound to a solid phase placed on the probe surface.  
     
     
         104 . The method of  claim 100  comprising preparing the population of biopolymers by chemical or enzymatic means to produce distinct analyte fragments of the biopolymer.  
     
     
         105 . The method of  claim 100  comprising preparing the population of biopolymers by cleaving monomers from the polymers chemically.  
     
     
         106 . The method of  claim 100  comprising preparing the population of biopolymers by cleaving monomers from the polymers enzymatically.  
     
     
         107 . The method of  claim 100  wherein the biopolymer is a nucleic acid.  
     
     
         108 . The method of  claim 100  wherein the biopolymer is a protein.  
     
     
         109 . The method of  claim 100  wherein the biopolymer is a polysaccharide.  
     
     
         110 . The method of  claim 100  wherein the probe comprises glass.  
     
     
         111 . The method of  claim 100  wherein the probe comprises ceramic.  
     
     
         112 . The method of  claim 100  wherein the probe comprises a synthetic polymer.  
     
     
         113 . The method of  claim 103  wherein the solid phase comprises a porous or non-porous bead of cross-linked polymer.  
     
     
         114 . The method of  claim 103  wherein the solid phase comprises a magnetic or paramagnetic material.  
     
     
         115 . The method of  claim 103  wherein the solid phase comprises a polymeric bead to which the affinity reagent is attached.  
     
     
         116 . The method of  claim 107  wherein the nucleic acids are modified by digestion with an exonuclease.  
     
     
         117 . The method of  claim 107  comprising cleavage of nucleic acids with endonuclease.  
     
     
         118 . The method of  claim 108  wherein the polypeptides are modified by exopeptidase digestion or N-terminal degradation by Edman degradation.  
     
     
         119 . The method of  claim 108  comprising cleavage of polypeptides with endoprotease or cyanogen bromide.  
     
     
         120 . The method of  claim 108  wherein the polypeptide is bound to a plurality of different photolabile attachment molecules and the method comprises determining the sequence of monomers in a plurality of fragments of the polypeptide, wherein the fragments are generated by endopeptidase digestion of the polypeptide.  
     
     
         121 . The method of  claim 109  wherein the polysaccharides are modified by digestion with neuraminidase, mannase, fucase, galactosidase, glucosidase, glyeanase or N-glycanase.  
     
     
         122 . The method of  claim 109  comprising cleavage of the polysaccharides with endoglycosidase H or endoglycosidase F.  
     
     
         123 . The method of  claim 109  wherein the polysaccharides are modified by digestion with FUCase I, MANase I, HEXase I, NANase III or PNGase.  
     
     
         124 . The method of  claim 113  wherein the cross-linked polymer is agarose, cellulose or dextran.