Separation devices and method for separating phosphorylated peptides and proteins

Abstract

Embodiments of the present invention are directed to articles of manufacture, devices, methods and apparatus for performing liquid chromatography featuring a chromatographic sorbent having one or more pentafluorophenyl groups, wherein said one or more pentafluorophenyl groups are a bonded phase on a sorbent selected from the group comprising silica, organic polymers or hybrid organic silane material and said pentafluorophenyl groups are in a mono-, bi-, and tridentate forms.

1 . A method for separating and isolating at least one phosphorylated peptide or phosporylated protein in a sample containing a mixture of peptides or proteins, comprising the steps of;
 (i) providing a mixed-mode chromatographic sorbent, a first eluent and a second eluent;   (ii) loading the sample onto mixed-mode chromatographic sorbent;   (iii) eluting from the mixed-mode chromatographic sorbent in a first mode using the first eluent to produce a first eluate;   (iv) eluting from the mixed mode chromatographic sorbent in a second mode using the second eluent to produce a second eluate; and,   (v) isolating at least one phosphorylated peptide or phosphorylated protein analyte in the first eluate or the second eluate.   
     
     
         2 . The method of  claim 1  where said chromatographic sorbent is capable of both reverse-phase and ion-exchange modes. 
     
     
         3 . The method of  claim 2  where said chromatographic sorbent is capable of both reverse-phase and cation-exchange modes. 
     
     
         4 . The method of  claim 2  where said chromatographic sorbent is capable of both reverse-phase and anion exchange modes. 
     
     
         5 . The method of  claim 4  where said chromatographic sorbent has one or more pentafluorophenyl groups. 
     
     
         6 . The method of  claim 5  wherein said one or more pentafluorophenyl groups are a bonded phase on a sorbent selected from the group comprising silica, organic polymers or hybrid organic silane material. 
     
     
         7 . The method of  claim 1  wherein said mixed mode sorbent surface conforms to the formula set forth below as Formula 1:
   (W 1 ) a (W 2 ) b (W 3 ) c Si(Z-C 6 F n H 5−n ) d    Formula 1   
       As used above, W 1 , W 2  and W 3  are independently selected from the groups consisting of hydrogen, hydroxyl, hydroxy-aliphatic, aliphatic, oxygen, nitrogen, and silane wherein at least one of, W 1 , W 2  and W 3  separately represent terminal silane valences bonded to oxygen, nitrogen, carbon or silane atoms of a support, Z represents a aliphatic moiety and n is an integer from one to 5 and a+b+c+d=4; where a is 1-3; b+c is less than or equal to 2, and d is less than or equal to 3. 
     
     
         8 . The method of  claim 7  wherein Z is —(CH 2 ) m — where m is 2 or 3. 
     
     
         9 . The method of  claim 7  wherein said support is silica where the terminal silane valences are bonded to silane. 
     
     
         10 . The method of  claim 7  wherein n is 5. 
     
     
         11 . The method of  claim 7  wherein said surface represents a distribution of the structure of Formula 1 with a distribution of monodentate, bidendate and tridentate forms. 
     
     
         12 . The method of  claim 11  wherein the support exhibits a distribution of monodentate, bidentate and tridentate forms, comprising 1 to 30 percent (1-30%) of the monodentate, 50 to 90 percent (50-90%) bidentate and 1 to 50 percent (1-50%) tridentate forms. 
     
     
         13 . The method of  claim 11  wherein the support exhibits a distribution of monodentate, bidentate and tridentate forms comprising 2 to about 25 percent of the monodentate, 60 to 80 percent (60-80%) of the bidentate and 10 to 40 percent (10-40%) tridentate forms. 
     
     
         14 . The method of  claim 1  where said chromatographic sorbent is held in a solid phase extraction device selected from the group consisting of columns, cartridges, well devices, and plates. 
     
     
         15 . The method of  claim 14  where said solid phase extraction device is part of a liquid chromatography system. 
     
     
         16 . The method of  claim 1  where said first eluent is a reverse-phase eluent. 
     
     
         17 . The method of  claim 1  where said second eluent contains a counterion for ion-exchange. 
     
     
         18 . The method of  claim 1  where said sample is a protein digest. 
     
     
         19 . The method of  claim 18  where said sample is a tryptic digest. 
     
     
         20 . The method of  claim 1  further comprising the step of flowing the sample through a solid phase extraction device to remove or separate analyte from other sample constituents. 
     
     
         21 . The method of  claim 20  wherein said solid phase extraction device is packed with particles having a metal oxide. 
     
     
         22 . The method of  claim 21  wherein said metal oxide is titanium or aluminium. 
     
     
         23 . The method of  claim 1  further comprising the step performing a liquid chromatography separation with at least some of first or second eluate to isolate one of more analytes. 
     
     
         24 . The method of  claim 23  where said step of liquid chromatography separation is by reverse-phase liquid chromatography. 
     
     
         25 . The method of  claim 1  where at least one of said analytes is identified by one of ultra-violet/visual spectroscopy, fourier transform ultra violet/visual spectroscopy, infra-red spectroscopy, fourier transform infra red spectroscopy, nuclear magnetic resonance spectroscopy, fourier transform nuclear magnetic resonance spectroscopy, raman spectroscopy, evaporative light scattering detection or mass spectrometry. 
     
     
         26 . A chromatographic sorbent having a surface conforming to the formula set forth below as Formula 1:
   (W 1 ) a (W 2 ) b (W 3 ) c Si(Z-C 6 F n H 5−n ) d    Formula 1   
       As used above, W 1 , W 2  and W 3  are independently selected from the groups consisting of hydrogen, hydroxyl, hydroxy-aliphatic, aliphatic, oxygen, nitrogen, and silane wherein at least one of, W 1 , W 2  and W 3  separately represent terminal silane valences bonded to oxygen, nitrogen, carbon or silane atoms of a support, Z represents a aliphatic moiety and n is an integer from one to 5 and a+b+c+d=4; where a is 1-3; b+c is less than or equal to 2, and d is less than or equal to 3. 
     
     
         27 . The chromatographic sorbent of  claim 26  wherein Z is —(CH 2 ) m — where m is 2 or 3. 
     
     
         28 . The chromatographic sorbent of  claim 26  wherein said support is silica where the terminal silane valences are bonded to silane. 
     
     
         29 . The chromatographic sorbent of  claim 26  wherein n is 5. 
     
     
         30 . The chromatographic sorbent of  claim 26  wherein said surface has a distribution of monodentate, bidendate and tridentate forms. 
     
     
         31 . The chromatographic sorbent of  claim 30  wherein said distribution of monodentate, bidentate and tridentate forms, comprising 1 to 30 percent (1-30%) of the monodentate, 50 to 90 percent (50-90%) bidentate and 1 to 50 percent (1-50%) tridentate forms. 
     
     
         32 . The chromatographic sorbent of  claim 30  wherein the support exhibits a distribution of monodentate, bidentate and tridentate forms comprising 2 to about 25 percent of the monodentate, 60 to 80 percent (60-80%) of the bidentate and 10 to 40 percent (10-40%) tridentate forms. 
     
     
         33 . The chromatographic sorbent of  claim 26  where said chromatographic sorbent is held in a solid phase extraction device selected from the group consisting of columns, cartridges, well devices, and plates. 
     
     
         34 . The chromatographic sorbent of  claim 33  where said solid phase extraction device is part of a liquid chromatography system. 
     
     
         35 . A device for performing liquid chromatography comprising in a solid phase extraction device selected from the group consisting of columns, cartridges, well devices, and plates having a chromatographic sorbent having a surface conforming to the formula set forth below as Formula 1:
   (W 1 ) a (W 2 ) b (W 3 ) c Si(Z-C 6 F n H 5−n ) d    Formula 1   
       As used above, W 1 , W 2  and W 3  are independently selected from the groups consisting of hydrogen, hydroxyl, hydroxy-aliphatic, aliphatic, oxygen, nitrogen, and silane wherein at least one of, W 1 , W 2  and W 3  separately represent terminal silane valences bonded to oxygen, nitrogen, carbon or silane atoms of a support, Z represents a aliphatic moiety and n is an integer from one to 5 and a+b+c+d=4; where a is 1-3; b+c is less than or equal to 2, and d is less than or equal to 3. 
     
     
         36 . The device of  claim 35  wherein Z is —(CH 2 ) m — where m is 2 or 3. 
     
     
         37 . The device of  claim 35  wherein n is 5. 
     
     
         38 . The device of  claim 35  wherein said support is silica where the terminal silane valences are bonded to silane. 
     
     
         39 . The device of  claim 35  wherein said surface has a distribution of monodentate, bidendate and tridentate forms. 
     
     
         40 . The device of  claim 39  wherein the support has a distribution of comprising 1 to 30 percent (1-30%) of the monodentate, 50 to 90 percent (50-90%) bidentate and 1 to 50 percent (1-50%) tridentate forms. 
     
     
         41 . The device of  claim 39  wherein the support has a distribution comprising 2 to about 25 percent of the monodentate, 60 to 80 percent (60-80%) of the bidentate and 10 to 40 percent (10-40%) tridentate forms. 
     
     
         42 . An apparatus for separating and isolating at least one phosphorylated peptide or phosphorylated protein in a sample containing a mixture of peptides or proteins and at least one analyte comprising a phosphorylated peptide or protein analysing a sample containing at least one analyte comprising;
 a solid phase extraction device selected from the group consisting of columns, cartridges, well devices, and plates, said solid phase extraction device having a mixed-mode chromatographic sorbent,   means for loading a sample onto said chromatographic sorbent of said solid phase extraction device,   means for introducing a first eluent to said chromatographic sorbent to produce a first eluate,   means for introducing a second eluent to said chromatographic sorbent to produce a second eluate,   wherein at least one phosphorylated peptide or phosphorylated protein analyte in the first eluate or the second eluate.   
     
     
         43 . The apparatus of  claim 42 , further comprising a means for collecting at least one of said first eluate and second eluate. 
     
     
         44 . The apparatus of  claim 42  where said chromatographic sorbent is capable of both reverse-phase and ion-exchange actions. 
     
     
         45 . The apparatus of  claim 42  where said chromatographic sorbent has having a surface conforming to the formula set forth below as Formula 1:
   (W 1 ) a (W 2 ) b (W 3 ) c Si(Z-C 6 F n H 5−n ) d    Formula 1   
       As used above, W 1 , W 2  and W 3  are independently selected from the groups consisting of hydrogen, hydroxyl, hydroxy-aliphatic, aliphatic, oxygen, nitrogen, and silane wherein at least one of, W 1 , W 2  and W 3  separately represent terminal silane valences bonded to oxygen, nitrogen, carbon or silane atoms of a support, Z represents a aliphatic moiety and n is an integer from one to 5 and a+b+c+d=4; where a is 1-3; b+c is less than or equal to 2, and d is less than or equal to 3. 
     
     
         46 . The device of  claim 45  wherein Z is —(CH 2 ) m — where m is 2 or 3. 
     
     
         47 . The device of  claim 45  wherein said support is silica where the terminal silane valences are bonded to silane. 
     
     
         48 . the device of  claim 45  wherein n is 5. 
     
     
         49 . The device of  claim 45  wherein said surface has a distribution of monodentate, bidendate and tridentate forms. 
     
     
         50 . The device of  claim 49  wherein said distribution comprises 1 to 30 percent (1-30%) of the monodentate, 50 to 90 percent (50-90%) bidentate and 1 to 50 percent (1-50%) tridentate forms. 
     
     
         51 . The device of  claim 49  wherein said distribution comprises 2 to about 25 percent of the monodentate, 60 to 80 percent (60-80%) of the bidentate and 10 to 40 percent (10-40%) tridentate forms. 
     
     
         52 . The apparatus of  claim 42  further comprising a means for further chromatographic separation of at least some of said first eluate or said second eluate. 
     
     
         53 . The apparatus of  claim 52  further comprising a means for further reverse-phase chromatographic separation of at least some of said first eluate or said second eluate. 
     
     
         54 . The apparatus of  claim 52  further comprising a secondary analyser downstream of said sorbent for analysing at least some of said first eluate or said second eluate. 
     
     
         55 . The apparatus of  claim 52  where said secondary analyser is an ultra-violet/visual spectroscope, fourier transform ultra violet/visual spectroscope, infra-red spectroscope, fourier transform infra red spectroscope, nuclear magnetic resonance spectroscope, fourier transform nuclear magnetic resonance spectroscope, raman spectroscope, evaporative light scattering detector or mass spectrometer. 
     
     
         56 . A method of making a a chromatographic sorbent having a surface conforming to the formula set forth below as Formula 1:
   (W 1 ) a (W 2 ) b (W 3 ) c Si(Z-C 6 F n H 5−n ) d    Formula 1   
       As used above, W 1 , W 2  and W 3  are independently selected from the groups consisting of hydrogen, hydroxyl, hydroxy-aliphatic, aliphatic, oxygen, nitrogen, and silane wherein at least one of, W 1 , W 2  and W 3  separately represent terminal silane valences bonded to oxygen, nitrogen, carbon or silane atoms of a support, Z represents a aliphatic moiety and n is an integer from one to 5 and a+b+c+d=4; where a is 1-3; b+c is less than or equal to 2, and d is less than or equal to 3, comprising the steps of reacting a silane surface with
   X a (W 2  ) b (W 3 ) c Si(Z-C 6 F n H 5−n ) d    Formula 2 
 
       As used above X is chloride, methoxy, ethoxy, alkyl-O, alkyne-O, hydroxyl, substituted amino group, trifluoromethanesulfonate or acid group and, W 2  and W 3  are independently selected from the group consisting of hydrogen, hydroxyl, hydroxy-alkyl of one to eight carbons, and hydroxy-alkene and hydroxy-alkyne of 2 to eight carbons, Z represents a alkyl chain of one to eight carbons or alkene or alkyne chain of 2 to eight carbons, and X 1 , X 2 , and X 3  separately represent terminal silane valences bonded to oxygen, nitrogen, carbon or silane atoms of a support, and n is an integer from one to about 1,000.