1. A method, comprising:
(a) providing a formalin-fixed, paraffin-embedded tissue sample comprising a plurality of targets;
(b) contacting the tissue sample with a first specific binding moiety capable of recognizing and binding to a first target in the tissue sample and a second specific binding moiety capable of recognizing and binding to a second target in the tissue sample;
(c) contacting the tissue sample with a first conjugate comprising a first enzyme and a first antibody capable of recognizing and binding to the first specific binding moiety;
(d) contacting the tissue sample with a first enzyme substrate moiety and separately a first mass tag precursor, wherein the first enzyme substrate moiety reacts with the first enzyme and the first mass tag precursor to produce and deposit a first mass tag at the first target;
(e) deactivating the first enzyme;
(f) contacting the tissue sample with a second conjugate comprising a second enzyme and a second antibody capable of recognizing and binding to the second specific binding moiety;
(g) contacting the tissue sample with a second enzyme substrate moiety and separately a second mass tag precursor, wherein the second enzyme substrate moiety reacts with the second enzyme and the second mass tag precursor to produce and deposit a second mass tag at the second target, wherein the first and second enzyme substrate moieties have the same chemical structure, the first and second mass tag precursors have the same chemical structure and at least one of the first mass tag precursor or second mass tag precursor is isotopically labeled such that the first mass tag and the second mass tag differ in mass;
(h) ionizing the first mass tag and the second mass tag to produce a first mass code and a second mass code, wherein each mass code has the same chemical structure but differs in mass from any other mass code produced from any other mass tag;
(i) detecting each mass code using a mass spectrometer; and
(j) quantifying relatively each target by quantifying an amount of each mass code by measuring a mass spectrometric peak having a mz ratio corresponding to an expected mz ratio of that mass code;
wherein the first mass tag precursor and the second mass tag precursor are selected from the group consisting of
where n is 1, 2, 3, 4, or 5 and wherein D is deuterium.
2. The method of claim 1, wherein the first mass tag precursor and the second mass tag precursor are selected from the group consisting of
3. The method of claim 1, wherein the first and second enzyme substrate moieties are selected from a methoxy naphthol or naphthol-azo compound and the first enzyme and the second enzymes are phosphatase enzymes.
4. The method of claim 1, further comprising:
contacting the tissue sample in step (b) with one or more subsequent specific binding moieties, each subsequent specific binding moiety capable of recognizing and binding to a subsequent target in the tissue sample;
deactivating the second enzyme after step (g);
sequentially performing the following steps for each of the one or more subsequent targets before performing step (h):
contacting the tissue sample with an additional conjugate for one of the one or more subsequent targets, the additional conjugate comprising an additional antibody and an additional enzyme, wherein the additional antibody is capable of recognizing and binding to the one of the one or more subsequent targets or to a specific binding moiety previously bound to the one of the one or more subsequent targets,
contacting the sample with an additional enzyme substrate moiety and an additional mass tag precursor, wherein the additional enzyme substrate moiety reacts with the additional enzyme and the additional mass tag precursor to produce and deposit an additional mass tag at the one of the one or more subsequent targets, wherein the additional enzyme substrate moiety has the same chemical structure as the first and second enzyme substrate moieties, the additional mass tag precursor has the same chemical structures as the first and second mass tag precursors, and the additional mass tag precursor is isotopically labeled such that the additional mass tag has a mass that differs from the masses of the first mass tag, the second mass tag, and any other additional mass tag; and
deactivating the additional enzyme if another subsequent target is present.
5. The method of claim 1, wherein, the first enzyme substrate moiety, the second enzyme substrate moiety, and any additional enzyme substrate moieties have the structure
and
the first enzyme, the second enzyme, and any additional enzymes are phosphatase enzymes.
6. The method of claim 1, wherein the plurality of targets comprises a plurality of cancer biomarkers.
7. The method of claim 6, wherein the cancer biomarkers comprise at least two breast cancer biomarkers selected from the group consisting of ER, PR, Her1, Her2, Her3, Her4, and Ki67.
8. The method of claim 6, wherein each cancer biomarker comprises a protein or a peptide, and wherein the first specific binding moiety is an antibody capable of recognizing and binding to a first cancer biomarker, the second specific binding moiety is an antibody capable of recognizing and binding to a second cancer biomarker, and each subsequent specific binding moiety is an antibody capable of recognizing and binding to a subsequent cancer biomarker.
9. The method of claim 6, wherein each cancer biomarker comprises a nucleic acid sequence, and wherein the first specific binding moiety is a labeled probe capable of recognizing and hybridizing to a first cancer biomarker or a portion thereof, the second specific binding moiety is a labeled probe capable of recognizing and hybridizing to a second cancer biomarker or a portion thereof, and each subsequent specific binding moiety is a labeled probe capable of recognizing and hybridizing to a subsequent cancer biomarker or a portion thereof.
10. The method of claim 1, wherein the first target comprises an intact form of a biomarker and the second target comprises one or more truncated forms of the biomarker.
11. The method of claim 10, wherein the first specific binding moiety is capable of recognizing and binding to the intact form of the biomarker, and the second specific binding moiety is capable of recognizing and binding to a truncated form of the biomarker and to the intact form of the biomarker, wherein the second enzyme substrate moiety reacts with the second enzyme and the second mass tag precursor to produce and deposit a second mass tag at the truncated form of the biomarker and at the intact form of the biomarker, and wherein quantifying the truncated form of the biomarker comprises quantifying the first mass code and the second mass code and determining the numerical difference between the quantified second mass code and the quantified first mass code.
12. A method, comprising:
(a) providing a formalin-fixed, paraffin-embedded tissue sample comprising a plurality of targets;
(b) contacting the tissue sample with a first specific binding moiety capable of recognizing and binding to a first target in the tissue sample and a second specific binding moiety capable of recognizing and binding to a second target in the tissue sample;
(c) contacting the tissue sample with a first conjugate comprising a first enzyme and a first antibody capable of recognizing and binding to the first specific binding moiety;
(d) contacting the tissue sample with a first enzyme substrate moiety and separately a first mass tag precursor, wherein the first enzyme substrate moiety reacts with the first enzyme and the first mass tag precursor to produce and deposit a first mass tag at the first target;
(e) deactivating the first enzyme;
(f) contacting the tissue sample with a second conjugate comprising a second enzyme and a second antibody capable of recognizing and binding to the second specific binding moiety;
(g) contacting the tissue sample with a second enzyme substrate moiety and separately a second mass tag precursor, wherein the second enzyme substrate moiety reacts with the second enzyme and the second mass tag precursor to produce and deposit a second mass tag at the second target, wherein the first and second enzyme substrate moieties have the same chemical structure, the first and second mass tag precursors have the same chemical structure and at least one of the first mass tag precursor or second mass tag precursor is isotopically labeled such that the first mass tag and the second mass tag differ in mass;
(h) ionizing the first mass tag and the second mass tag to produce a first mass code and a second mass code, wherein each mass code has the same chemical structure but differs in mass from any other mass code produced from any other mass tag;
(i) detecting each mass code using a mass spectrometer; and
(j) quantifying relatively each target by quantifying an amount of each mass code by measuring a mass spectrometric peak having a mz ratio corresponding to an expected mz ratio of that mass code;
wherein the tissue sample comprises from 2 to 4 targets, and the first mass tag precursor, the second mass tag precursor are selected from the group consisting of
wherein D is deuterium and n is 1, 2, 3, 4, or 5.
13. The method of claim 12, wherein the first mass tag precursor, the second mass tag precursor, and any additional mass tag precursors are selected from the group consisting of
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. An armrest for a vehicle passenger compartment comprising:
a collapsible frame deflectable in at least one direction upon a forcible impact received by the armrest, wherein the collapsible frame comprises:
a first frame member adapted to be mounted to a support member in the vehicle passenger compartment to mount the armrest to the support member; and
a second frame member mounted to the first frame member by a moveable mounting, wherein the moveable mounting comprises a mounting adapted to permit movement of the second frame member relative to the first frame member upon receipt of the forcible impact to the armrest, and at least one pin on one of the first frame member and the second frame member received by at least one aperture on the other of the first frame member and the second frame member corresponding to each of the at least one pin on the one of the first frame member and the second frame member, wherein the at least one pin is sheared off by the at least one corresponding aperture during relative movement of the second frame member with respect to the first frame member;
a recess located within the armrest adjacent to the second frame member, wherein the recess provides an area for deflection of the second frame member upon receipt of the forcible impact; and
a cover positioned over the collapsible frame.
2. The armrest of claim 1 wherein the forcible impact required to move the second frame member is less than that required if the second frame member were permanently mounted to the first frame member.
3. The armrest of claim 1 wherein the forcible impact required to move the second frame member is less than that able to cause injury to a bodily region of an occupant that collides with the armrest.
4. The armrest of claim 1 wherein the at least one aperture has a frustoconical shape to assist in shearing off the at least one pin.
5. The armrest of claim 1 and further comprising a core material located between the cover and the collapsible frame to provide a tactile feel for the armrest.
6. The armrest of claim 5 wherein the core is composed of a foam material.
7. The armrest of claim 6 wherein the foam material is semi-rigid.
8. The armrest of claim 7 wherein the cover is composed of a flexible material.
9. The armrest of claim 8 wherein the cover is composed of one of a leather, a fabric, and a polymeric material.
10. The armrest of claim 9 wherein the cover provides an architectural finish and is composed of a material that matches the vehicle passenger compartment.
11. The armrest of claim 10 wherein a forcible impact received against the cover deflects at least a portion of the core against the collapsible frame to cause deflection of the collapsible frame.
12. The armrest of claim 11 wherein the forcible impact required to move the second frame member is less than that required if the second frame member were permanently mounted to the first frame member.
13. The armrest of claim 11 wherein the forcible impact required to move the second frame member is less than that able to cause injury to a bodily region of an occupant that collides with the armrest.
14. The armrest of claim 3 and further comprising a core material located between the cover and the collapsible frame to provide a tactile feel for the armrest.
15. The armrest of claim 14 wherein the core is composed of a foam material.
16. The armrest of claim 15 wherein the foam material is semi-rigid.
17. The armrest of claim 14 wherein a forcible impact received against the cover deflects at least a portion of the core against the collapsible frame to cause deflection of the collapsible frame.
18. The armrest of claim 1 wherein the cover is composed of a flexible material.
19. The armrest of claim 1 wherein the cover is composed of one of a leather, a fabric, and a polymeric material.
20. The armrest of claim 1 wherein the cover provides an architectural finish and is composed of a material that matches the vehicle passenger compartment.
21. An armrest for a vehicle passenger compartment comprising:
a substrate adapted to be fixedly mounting to a support panel in the vehicle passenger compartment;
a cover mounted over the substrate;
a core disposed between at least a portion of the substrate and the cover to provide a tactile feel to the armrest;
a deflectable member retained against the substrate, wherein the deflectable member can overcome its retention against the substrate upon receipt of a suitably large impact force and move with respect to the substrate; and
a moveable mounting adapted to permit movement of the deflectable member relative to the substrate upon receipt of a suitably large impact force to the armrest, wherein the retention of the deflectable member is performed by the moveable mounting;
wherein the moveable mounting comprises at least one pin on one of the substrate and the deflectable member received by at least one aperture on the other of the substrate and the deflectable member corresponding to each of the at least one pin on the one of the substrate and the deflectable member; and
wherein the at least one pin is sheared off by the at least one corresponding aperture when the deflectable member overcomes its retention against the substrate upon receipt of the impact force.
22. The armrest of claim 21 and further comprising a recess adjacent to the substrate, wherein the deflectable member can move into an area of the recess upon receipt of the impact force.
23. The armrest of claim 22 wherein the impact force deflects at least a portion of the core against the deflectable member to cause movement of the deflectable member.
24. The armrest of claim 21 wherein the at least one aperture has a frustoconical shape to assist in shearing off the at least one pin.
25. The armrest of claim 21 wherein the movable mounting comprises at least one edge on one of the deflectable member and the substrate received behind at least one ridge on the other of the deflectable member and the substrate corresponding to each of the at least one edge on the one of the deflectable member and the substrate.
26. The armrest of claim 25 wherein the at least one edge cams over the at least one ridge when the deflectable member overcomes its retention against the substrate upon receipt of the impact force.
27. The armrest of claim 26 wherein the at least one edge has one of a semi-circular and triangular profile.
28. The armrest of claim 27 wherein the cover is composed of a flexible material.
29. The armrest of claim 28 wherein the cover is composed of one of a leather, a fabric, and a polymeric material.
30. The armrest of claim 29 wherein the cover provides an architectural finish and is composed of a material that matches the vehicle passenger compartment.
31. The armrest of claim 30 wherein the core is composed of a foam material.
32. The armrest of claim 31 wherein the foam material is semi-rigid.
33. The armrest of claim 32 wherein the impact force required to move the deflectable member is less than that required if the deflectable member were permanently mounted to the substrate.
34. The armrest of claim 32 wherein the impact force required to move the deflectable member is less than that able to cause injury to a bodily region of an occupant that collides with the armrest.
35. The armrest of claim 21 wherein the impact force deflects at least a portion of the core against the deflectable member to cause movement of the deflectable member.
36. The armrest of claim 21 wherein the impact force required to move the deflectable member is less than that able to cause injury to a bodily region of an occupant that collides with the armrest.
37. The armrest of claim 21 wherein the cover is composed of a flexible material.
38. The armrest of claim 21 wherein the cover is composed of one of a leather, a fabric, and a polymeric material.
39. The armrest of claim 21 wherein the cover provides an architectural finish and is composed of a material that matches the vehicle passenger compartment.
40. The armrest of claim 21 wherein the core is composed of a foam material.
41. The armrest of claim 40 wherein the foam material is semi-rigid.
42. An armrest for a vehicle passenger compartment comprising:
a substrate adapted to be fixedly mounted to a support panel in the vehicle passenger compartment, the substrate defining a recess adjacent to the support panel and at least one retainer in register with the recess, the at least one retainer spaced inwardly from the support panel to define a translation zone between the support panel and the at least one retainer;
a deflectable member located in the recess for movement relative to the substrate, wherein the deflectable member has at least one engagement portion engaged with each of the at least one retainer on the substrate, wherein the engagement of the at least one retainer with the at least one engagement portion retains the deflectable member from moving into the translation zone, and wherein, upon receipt of a sufficiently large impact to the arm rest, the engagement of the at least one engagement portion with the at least one retainer is overcome and the deflectable member travels into the translation zone relative to the substrate;
a cover mounted over the substrate; and
a core disposed between at least a portion of the substrate and the cover to provide a tactile feel to the armrest.
43. The armrest of claim 42 wherein the retainer comprises at least one shear pin on one of the substrate and the deflectable member received by at least one aperture on the other of the substrate and the deflectable member corresponding to each of the at least one shear pin on the one of the substrate and the deflectable member.
44. The armrest of claim 43 wherein the at least one shear pin is sheared off by the at least one corresponding aperture when the deflectable member overcomes its retention against the substrate upon receipt of the impact.
45. The armrest of claim 44 wherein the at least one aperture has a frustoconical shape to assist in shearing off the at least one shear pin.
46. The armrest of claim 42 wherein the movable mounting comprises at least one edge on one of the deflectable member and the substrate received behind at least one ridge on the other of the deflectable member and the substrate corresponding to each of the at least one edge on the one of the deflectable member and the substrate.
47. The armrest of claim 46 wherein the at least one edge cams over the at least one ridge when the deflectable member overcomes its retention against the substrate upon receipt of the impact.
48. The armrest of claim 47 wherein the at least one edge has one of a semi-circular and triangular profile.
49. The armrest of claim 42 wherein the cover is composed of a flexible material.
50. The armrest of claim 49 wherein the cover is composed of one of a leather, a fabric, and a polymeric material.
51. The armrest of claim 50 wherein the cover provides an architectural finish and is composed of a material that matches the vehicle passenger compartment.
52. The armrest of claim 42 wherein the core is composed of a foam material.
53. The armrest of claim 52 wherein the foam material is semi-rigid.
54. The armrest of claim 42 wherein the impact required to move the deflectable member is less than that required if the deflectable member were permanently mounted to the substrate.
55. The armrest of claim 42 wherein the impact required to displace the deflectable member is less than that able to cause injury to a bodily region of an occupant that collides with the armrest.
56. The armrest of claim 42 wherein the impact deflects at least a portion of the core against the deflectable member to cause displacement of the deflectable member.