Cationic Tags for Attomole Level Detection of Analytes by Mass Spectrometry

A method for detecting an analyte A using a multi-stage mass spectrometer having at least first and second stages, the method comprising the steps of:

• (a) contacting a sample containing the analyte with a molecular tag Q+, whereby the analyte is covalently labeled with the molecular tag to produce tagged analyte Q+-A;
• wherein Q+ is represented by Formula (I)
• wherein R1, R2, and R3 are each independently selected from methyl, ethyl, propyl, cyclopropyl, isopropyl, butyl, fluoromethyl, difluoromethyl, trifluoromethyl, fluoroethyl, difluoroethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, and nitroethyl, and each E is independently hydrogen or deuterium;
• wherein one of R4-R8 bears a reactivity group that enables Q+ to be attached covalently to the analyte to form Q+-A;
• wherein the remainder of R4-R8 are independently selected from H, D, CE2COCE3, CE2CONE2, CE2NHCOCE3, CE2OG, CE2SG, OG, and SG, wherein G is methyl, ethyl, or propyl bearing H or D atoms, or a combination thereof;
• wherein each CE2COCE3, CE2CONE2, CE2NHCOCE3, CE2OG, CE2SG, OG and SG is ortho or para to the CE2NR1R2R3 substituent;
• wherein Q+-A comprises at least one O, N or S atom, which is separated from the C atom of the CE2NR1R2R3 substituent by four or five single or double bonds, and said O, N, or S atom is in a group which is ortho to the CE2NR1R2R3 substituent;
• (b) subjecting Q+-A to volatization into the gas phase and then filtration in the first stage;
• (c) subjecting Q+-A to cleavage by energetic activation to form a resonance-stabilized first product ion q+-A and a neutral amine, wherein q+ and the neutral amine are both fragments of Q+;
• (d) subjecting q+-A to cleavage by energetic activation to form a second product ion q+-A′, wherein A′ is a fragment of A;
• and
• (e) detecting q+-A′.

A method for detecting an analyte A using a multi-stage mass spectrometer having at least first and second stages, the method comprising the steps of:

• (a) contacting a sample containing the analyte with a molecular tag Q+, whereby the analyte is covalently labeled with the molecular tag to produce tagged analyte Q+-A;
• wherein Q+ is represented by Formula (II)
• wherein Z+ is selected from pyridinium, fluorine-substituted pyridinium, methoxy-substituted pyridinium, quinolinium, or triphenylphosphonium group, and each E is independently hydrogen or deuterium;
• wherein one of R4-R8 bears a reactivity group that enables Q+ to be attached covalently to the analyte to form Q+-A;
• wherein the remainder of R4-R8 are independently selected from H, D, CE2COCE3, CE2CONE2, CE2NHCOCE3, CE2OG, CE2SG, OG, and SG, wherein G is methyl, ethyl, or propyl bearing H or D atoms, or a combination thereof;
• wherein each CE2COCE3, CE2CONE2, CE2NHCOCE3, CE2OG, CE2SG, OG and SG is ortho or para to the CE2Z+ substituent;
• wherein Q+-A comprises at least one O, N or S atom, which is separated from the C atom of the CE2Z+ substituent by four or five single or double bonds, and said O, N, or S atom is in a group which is ortho to the CE2Z substituent;
• (b) subjecting Q+-A to volatization into the gas phase and then filtration in the first stage;
• (c) subjecting Q+-A to cleavage by energetic activation to form a resonance-stabilized first product ion q+-A and a neutral amine, wherein q+ and the neutral amine are both fragments of Q+;
• and
• (d) detecting q+-A.

A method for detecting an analyte A using a multi-stage mass spectrometer having at least first and second stages, the method comprising the steps of:

• (a) contacting a sample containing the analyte with a molecular tag Q+, whereby the analyte is covalently labeled with the molecular tag to produce tagged analyte Q+-A;
• wherein Q+ is represented by Formula (II)
• wherein Z+ is selected from pyridinium, fluorine-substituted pyridinium, methoxy-substituted pyridinium, quinolinium, or triphenylphosphonium group, and each E is independently hydrogen or deuterium;
• wherein one of R4-R8 bears a reactivity group that enables Q+ to be attached covalently to the analyte to form Q+-A;
• wherein the remainder of R4-R8 are independently selected from H, D, CE2COCE3, CE2CONE2, CE2NHCOCE3, CE2OG, CE2SG, OG, and SG, wherein G is methyl, ethyl, or propyl bearing H or D atoms, or a combination thereof;
• wherein each CE2COCE3, CE2CONE2, CE2NHCOCE3, CE2OG, CE2SG, OG and SG is ortho or para to the CE2Z+ substituent;
• wherein Q+-A comprises at least one O, N or S atom, which is separated from the C atom of the CE2Z+ substituent by four or five single or double bonds, and the O, N, or S atom is in a group which is ortho to the CE2Z substituent;
• (b) subjecting Q+-A to volatization into the gas phase and then filtration in the first stage;
• (c) subjecting Q+-A to cleavage by energetic activation to form a resonance-stabilized first product ion q+-A and a neutral amine, wherein q+ and the neutral amine are both fragments of Q+;
• (d) subjecting q+-A to cleavage by energetic activation to form a second product ion q+-A′, wherein A′ is a fragment of A;
• and
• (e) detecting q+-A′.

A molecular tag for identification of an analyte by multi-stage mass spectrometry, the molecular tag having a structure according to Formula (I)

• wherein R1, R2, and R3 are each independently selected from methyl, ethyl, propyl, cyclopropyl, isopropyl, butyl, fluoromethyl, difluoromethyl, trifluoromethyl, fluoroethyl, difluoroethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, and nitroethyl, and each E is independently hydrogen or deuterium;
• wherein R4 bears a reactivity group that enables the molecular tag to be attached covalently to an analyte;
• and
• wherein the remainder of R5-R8 are independently selected from H, D, CE2COCE3, CE2CONE2, CE2NHCOCE3, CE2OG, CE2SG, OG, and SG, wherein G is methyl, ethyl, or propyl bearing H or D atoms, or a combination thereof;
• wherein each CE2COCE3, CE2CONE2, CE2NHCOCE3, CE2OG, CE2SG, OG and SG is ortho or para to the CE2NR1R2R3 substituent.

A molecular tag for identification of an analyte by multi-stage mass spectrometry, the molecular tag having a structure according to Formula (II):

• wherein Z+ is selected from pyridinium, fluorine-substituted pyridinium, methoxy-substituted pyridinium, quinolinium, or triphenylphosphonium group, and each E is independently hydrogen or deuterium;
• wherein R4 bears a reactivity group that enables the molecular tag to be attached covalently to an analyte;
• and
• wherein the remainder of R5-R8 are independently selected from H, D, CE2COCE3, CE2CONE2, CE2NHCOCE3, CE2OG, CE2SG, OG, and SG, wherein G is methyl, ethyl, or propyl bearing H or D atoms, or a combination thereof;
• wherein each CE2COCE3, CE2CONE2, CE2NHCOCE3, CE2OG, CE2SG, OG and SG is ortho or para to the CE2Z+ substituent.