BACKGROUND OF INVENTION

Proteome quantification has become an increasingly essential component of modern biology and translational medicine. Whether targeted or global, stable isotope incorporation with mass spectrometry (MS) analysis is the primary mechanism by which protein abundance measurements are determined. There are numerous approaches to introduce stable isotopes into peptides—SILAC, isobaric tagging (TMT/iTRAQ), iCAT, etc. In most conventional approaches, however, these methods incorporate heavy isotopes to increase mass by at least 1 Da. SILAC, the quantification gold standard, for example, typically utilizes a 4 Da spacing so as to limit the isotopic cluster overlap of the heavy and light peptides. This requirement limits the quantitative capacity of SILAC to triplex. The reason for this is twofold: (1) the mass of the amino acids can only be elevated to ˜+12 Da and (2) mass spectral complexity is increased as multiple isotopic clusters are introduced.

Isobaric tagging addresses the problem of increases in mass spectra complexity by concealing the quantitative information in the MS1scan, thereby permitting a higher level of multiplexing than obtained via conventional SILAC methods. McAlister et al. recently report methods for expanding the throughput of methods using TMT isobaric reagents from 6-plex to 8 plex, for example, via techniques that resolve the relatively small isotopic shift resulting from substitution of a15N for a13C in the isobaric tagging agents. [See, Mc Alister et al.,Analytical Chemistry, accepted manuscript, DOI:10.1021/ac301572t]. Despite the advances in the degree of multiplexing accessible using isobaric tagging, these methods have been demonstrated to be susceptible to certain limitations that impact their use in quantitative analysis for applications in proteomics. First, isobaric methods suffer from severe dynamic range compression and loss of quantitative accuracy due to precursor interference with in the MS/MS isolation window. Precursor interference in isobaric methods, for example, has been demonstrated to significantly degrade the quantitative accuracy of the technique. Second, quantitative data can only be obtained for peptides that are selected for further MS2analysis When replicate analyses are necessary, therefore, this becomes a serious problem as there is high variation in which peptides are selected for MS2from one run to the next (˜60%). Third, current isobaric tagging methods are only compatible with collisional activation for dissociation, thus limiting the overall versatility of this technique.

From the foregoing it shall be apparent that a need currently exists for mass spectrometry techniques for proteomic analysis. For example, advanced mass spectrometry techniques are needed that are capable of achieving high degrees of multiplexing necessary for high throughput analysis of protein containing samples. In addition, advanced mass spectrometry techniques are needed that are not susceptible to problems of precursor interference that can impact quantitative accuracy and that are compatible with a range of dissociation techniques including electron capture and electron transfer dissociation methods.

SUMMARY OF THE INVENTION

The invention provides mass spectrometry methods, compositions and systems which enable a unique platform for analyte quantitation accessing very high degrees of multiplexing and accurate quantification, particularly well-suited for a range of quantitative analysis for proteomics applications. Embodiments of the present methods and systems combine isotopic coding agents characterized by very small differences in molecular mass with mass spectrometry methods providing large resolving power to provide relative or absolute analyte quantification in a large number of samples. In some embodiments, for example, quantification methods of the invention access a high degree of multiplexing by introducing isotopic labels from a large number of (e.g., ranging from 2 to 10 and in some embodiments greater than 20) isotopic coding agents that are isotopologues, such as amino acids, tagging agents and/or synthetic proteins and peptides, having mass differences that can be accurately resolved using high resolution mass spectrometry. The methods, compositions and systems described herein enable increased quantitation accuracy compatible with multiplexing necessary to achieve high levels of throughput.

In an aspect, the invention provides a method for determining the abundances of an analyte in a plurality of samples comprising the steps of: (a) providing a plurality of cell cultures including at least a first cell culture and a second cell culture; (b) providing a different isotopically labeled amino acid to each of the cell cultures, wherein the isotopically labeled amino acids of each of the cell cultures are isotopologues of the same amino acid; (c) growing cells of each of the cell cultures, thereby introducing a different isotopic label into proteins generated by each cell culture; (d) generating a sample for each of the cell cultures, wherein each sample is characterized by a different isotopically labeled analyte, the samples including at least a first sample for the first cell culture having a first isotopically labeled analyte and a second sample for the second cell culture having a second isotopically labeled analyte, wherein the isotopically labeled analytes of each sample are isotopologues; and wherein the difference of the molecular masses of the first isotopically labeled analyte and the second isotopically labeled analyte is less than or equal to 300 mDa; (e) analyzing the isotopically labeled analytes for each sample using a mass spectrometry analysis technique providing a resolving power equal to or greater than 100,000, thereby generating an independent and distinguishable mass spectrometry signal for the isotopically labeled analytes of each sample; and (f) comparing the mass spectrometry signals for the isotopically labeled analytes of each sample, thereby determining the abundances of the analyte in the plurality of samples.

In an aspect, the invention provides a method for determining the abundances of an analyte in a plurality of samples comprising the steps of: (a) providing the plurality of samples each having the analyte including at least a first sample and a second sample; (b) providing a different isotopic tagging reagent to each sample, wherein the isotopic tagging reagents of each of the samples are isotopologues, and wherein the isotopic tagging reagents are not isobaric tags having a reporter group and a mass balancing group; (c) chemically reacting the analyte and isotopic tagging reagent of each sample, thereby generating a different isotopically labeled analyte for each sample including a first isotopically labeled analyte for the first sample and a second isotopically labeled analyte for the second sample; wherein the isotopically labeled analytes of each sample are isotopologues; and wherein the difference of the molecular masses of the first isotopically labeled analyte and the second isotopically labeled analyte is less than or equal to 300 mDa; (d) analyzing the isotopically labeled analytes for each sample using a mass spectrometry analysis technique providing a resolving power equal to or greater than 100,000, thereby generating an independent and distinguishable mass spectrometry signal for the isotopically labeled analytes of each sample; and (e) comparing the mass spectrometry signals for the isotopically labeled analytes of each sample, thereby determining the abundance of the analyte in the plurality of samples.

In an aspect, the invention provides a method for determining the abundances of an analyte in a plurality of samples comprising the steps of: (a) providing the plurality of samples each having the analyte including at least a first sample and a second sample; (b) providing a different isotopic tagging reagent to each sample, wherein the isotopic tagging reagents of each of the samples are isotopologues; (c) chemically reacting the analyte and isotopic tagging reagent of each sample, thereby generating a different isotopically labeled analyte for each sample including a first isotopically labeled analyte for the first sample and a second isotopically labeled analyte for the second sample; wherein the isotopically labeled analytes of each sample are isotopologues; and wherein the difference of the molecular masses of the first isotopically labeled analyte and the second isotopically labeled analyte is less than or equal to 300 mDa; (d) analyzing the isotopically labeled analytes for each sample using a mass spectrometry analysis technique providing a resolving power equal to or greater than 100,000, thereby generating an independent and distinguishable mass spectrometry signal for the isotopically labeled analytes of each sample; and (e) comparing the mass spectrometry signals for the isotopically labeled analytes of each sample, thereby determining the abundance of the analyte in the plurality of samples; wherein the step of analyzing the isotopically labeled analytes for each sample using a mass spectrometry analysis technique does not use an isobaric tagging method, for example, wherein the step of analyzing the isotopically labeled analytes for each sample using a mass spectrometry analysis technique does not generate a reporter ion or mass spectrometry data corresponding to a reporter ion.

In some methods of the invention, the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins of the samples do not comprise an isobaric mass tag, such as an TMT or iTRAQ mass tag. In some embodiments, for example, the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins of the samples do not have at least a portion of the functional groups of conventional isobaric mass tags, such as not having a reporter group and/or not having a mass balancing group. It is noted, however, that isotopic tagging reagents of the invention commonly containing reactive groups, such as protein or peptide reactive groups, for example, to allow incorporation of an isotopic label into the analyte via chemical reactions.

In some methods of the invention, the step of analyzing the isotopically labeled analytes for each sample is carried out using a single stage mass spectrometry technique, such as a technique involving fragmentation and detection of product ions generated directed from the analyte such as ions generated directly from electrospray ionization and MALDI techniques. In some embodiments, for example, the step of analyzing the isotopically labeled analytes for each sample using the mass spectrometry analysis technique comprises: (i) generating ions from each of the isotopically labeled analytes for each sample; (ii) fragmenting the ions so as to generate product ions having a different isotopic label for each sample; and (iii) detecting the product ions for each sample. In some embodiments, for example, the product ions are peptide fragment ions having the isotopic label, optionally wherein the product ions are detected without further mass selection or fragmentation of the product ions. In a specific embodiment, the step of analyzing the isotopically labeled analytes for each sample is not carried out using a MSxmultiple stage mass spectrometry, wherein x is greater than or equal to 2, for example, wherein the step of analyzing the isotopically labeled analytes for each sample is not carried out using tandem mass spectrometry. Alternatively, the invention includes methods wherein the step of analyzing the isotopically labeled analytes for each sample is carried out using a MSxmultiple stage mass spectrometry, wherein x is greater than or equal to 2, such as tandem mass spectrometry techniques.

Methods of the invention are compatible with a broad range of approaches for introducing isotopic labels into analytes for generating isotopically labeled analytes, such as reactive techniques, synthetic techniques and metabolic techniques. In reactive techniques, for example, one or more isotopic tagging reagents are provided to a sample under conditions (e.g., concentration of tagging reagent, temperature, pH, ionic strength, solvent composition, etc.) such that at least a portion of the isotopic tagging reagent reacts with analyte to generate isotopically labeled analyte. In synthetic techniques, for example, one or more isotopically labeled standards, such as an isotopically labeled peptide standard, is synthesized, for example via chemical reaction(s) of isotope encoded amino acids, and then added to a sample under analysis. In metabolic techniques, for example, isotope encoded compounds, such as isotopically labeled amino acids or peptides, are provided to a cell culture under conditions wherein the isotopically labeled amino acids or peptides are incorporated into peptides and modified peptides generated by the cells.

In an embodiment, for example, the step of providing the different isotopically labeled amino acids to each of the cell cultures comprises providing a growth medium to each of the cell cultures comprising the isotopically labeled amino acids. In an embodiment, for example, the introducing a different isotopic label into proteins generated by each cell culture is achieved via metabolic incorporation of the isotopically labeled amino acids into cells of the cell cultures. In an embodiment, for example, the step of generating a sample for each of the cell cultures comprises lysing the cells of each of the cell cultures. In an embodiment, for example, the step of generating a sample for each of the cell cultures comprises extracting proteins of each of the cell cultures. In an embodiment, for example, the step of generating a sample for each of the cell cultures comprises digesting proteins of each of the cell cultures. In an embodiment, for example, the samples are digested using trypsin or Endo LysC.

An important aspect of the present methods is use of a series of isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins having differences in mass that can be resolved using a mass spectrometry analysis technique providing a resolving power equal to or greater than 100,000. Use of at least a portion of the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins having small differences in molecular mass (e.g., less than or equal to 300 mDa) is beneficial in some embodiments for accessing high multiplexing capabilities. In some embodiments, for example, the step of analyzing the isotopically labeled analytes for each sample comprises resolving differences of the mass to charge ratios and/or molecular masses of the isotopically labeled analytes. In some embodiments, for example, the difference of the molecular masses of the first isotopically labeled analyte and the second isotopically labeled analyte is less than or equal to 100 mDa, and optionally for some applications wherein the difference of the molecular masses of the first isotopically labeled analyte and the second isotopically labeled analyte is less than or equal to 50 mDa and optionally for some applications wherein the difference of the molecular masses of the first isotopically labeled analyte and the second isotopically labeled analyte is greater than or equal to 10 mDa. In some embodiments, for example, the difference of the molecular masses of the first isotopically labeled analyte and the second isotopically labeled analyte is selected over the range of 100 mDa to 1 mDa, and optionally for some applications wherein the difference of the molecular masses of the first isotopically labeled analyte and the second isotopically labeled analyte is selected over the range of 50 mDa to 1 mDa, and optionally for some applications wherein the difference of the molecular masses of the first isotopically labeled analyte and the second isotopically labeled analyte is selected over the range of 10 mDa to 1 mDa. In some embodiments, for example, each of the isotopically labeled analytes have a molecular mass within 100 mDa to 1 mDa of another of the isotopically labeled analyte, and optionally for some applications each of the isotopically labeled analytes have a molecular mass within 50 mDa to 1 mDa of another of the isotopically labeled analyte and optionally for some applications each of the isotopically labeled analytes have a molecular mass within 10 mDa to 1 mDa of another of the isotopically labeled analyte. In some embodiments, for example, the molecular masses of each of the isotopically labeled analytes are within a range of 10000 mDa to 10 mDa, and optionally for some applications the molecular masses of each of the isotopically labeled analytes are within a range of 1000 mDa to 10 mDa, and optionally for some applications the molecular masses of each of the isotopically labeled analytes are within a range of 100 mDa to 10 mDa.

Different isotopically encoded compounds of the invention can have a number of stable heavy isotopes selected over a wide range for different applications. As used herein isotopically encoded compounds refers to compound having one or more stable heavy isotopes functioning as an isotopic label. Isotopically encoded compounds include a range of tagging reagents, standards and/or labeled analytes, such as isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids, isotopically labeled standards and/or isotopically labeled peptide or proteins. Isotopically encoded compounds include compounds having one or more stable heavy isotopes that are isotopologues, for example, isotopologues that can be accurately distinguished using mass spectrometry based on measured mass-to-charge ratios.

In an embodiment, for example, the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins have a number of stable heavy isotopes selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. In an embodiment, for example, the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins have a number of stable heavy isotopes equal to or greater than 1, and optionally for some applications a number of stable heavy isotopes equal to or greater than 4, and optionally for some applications a number of stable heavy isotopes equal to or greater than 10, and optionally for some applications a number of stable heavy isotopes equal to or greater than 15.

In some embodiments, for example, the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins are selected from the group consisting of: an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one15N isotope; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one13C isotope; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one18O isotope; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one34S isotope; and an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one2H isotope.

In some embodiments, for example, the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins are selected from the group consisting of: an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least two13C isotopes; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one13C isotope and at least one15N isotope; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one13C isotope and at least one2H isotope; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one13C isotope and at least one18O isotope; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one13C isotope and a34S isotope; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least two15N isotopes; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one15N isotope and at least one2H isotope; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one15N isotope and at least one18O isotope; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one15N isotope and at least one34S isotope; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least two2H isotopes; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one2H isotope and at least one18O isotope; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one2H isotope and at least one34S isotope; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least two18O isotopes; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one18O isotope and at least one34S isotope; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one13C isotope, at least one15N isotope and at least one2H isotope; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one13C isotope, at least one15N isotope and at least one18O isotope; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one13C isotope, at least one15N isotope and at least one34S isotope; and an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having at least one18O isotope, at least one15N isotope and at least one34S isotope.

In an embodiment, for example, the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins are selected from the group consisting of: an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein isotopically labeled amino acid selected from the group consisting of: an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having 1, 2, 3, or 415N isotopes; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1113C isotopes; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having 1 or 218O isotopes; an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having one34S isotope; and an isotopically labeled analyte, isotopic tagging reagent, isotopically labeled amino acid and/or isotopically labeled peptide or protein having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 102H isotopes.

Methods of the invention include quantification approaches using isotopically encoded amino acids, such as isotopically labeled amino acids. In an embodiment, for example, the isotopically labeled amino acids are isotopologues of a naturally occurring amino acid. In an embodiment, for example, the isotopically labeled amino acids are isotopologues of serine, leucine, tyrosine, lysine, methionine, or arginine. In an embodiment, for example, the isotopologues have a number of stable heavy isotopes selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. In an embodiment, for example, the isotopically labeled amino acids of each sample have an isotopic composition for its coded element formula selected from the group consisting of:

12C3-i13Ci1H4-j2Hj14N1-n15Nn16O1-o18Oo, wherein i≤3, j≤4, n≤1, o≤1;

12C6-i13Ci1H7-j2Hj14N4-n15Nn16O1-o18Oo, wherein i≤6, j≤7, n≤4, o≤1;

12C4-i13Ci1H3-j2Hj14N2-n15Nn16O2-o18Oo, wherein i≤4, j≤3, n≤2, o≤2;

12C4-i13Ci1H3-j2Hj14N1-n15Nn16O2-o18Oo, wherein i≤4, j≤3, n≤1, o≤2;

12C3-i13Ci1H3-j2Hj14N1-n15Nn16O1-o18Oo32S1-p34Sp, wherein i≤3, j≤3, n≤1, o≤1, p≤1;

12C5-i13Ci1H5-j2Hj14N1-n15Nn16O2-o18Oo, wherein i≤5, j≥5, n≤18, o≤2;

12C5-i13Ci1H5-j2Hj14N2-n15Nn16O2-o18Oo, wherein i≤5, j≤5, n≤2, o≤2;

12C2-i13Ci1H2-j2Hj14N1-n15Nn16O1-o18Oo, wherein i≤2, j≤2, n≤1, o≤8;

12C6-i13Ci1H5-j2Hj14N3-n15N3-n15Nn16O1-o18Oo, wherein i≤6, j≤5, n≤3, o≤2;

12C6-i13Ci1H10-j2Hj14N1-n15Nn16O1-o18Oo, wherein i≤6, j≤10, n≤1, o≤1;

12C6-i13Ci1H10-j2Hj14N1-n15Nn16O1-o18Oo, wherein i≤6, j≤10, n≤1, o≤1;

12C6-i13Ci1H9-j2Hj14N2-n15Nn16O1-o18Oo, wherein i≤6, j≤9, n≤2, o≤1;

12C5-i13Ci1H8-j2Hj14N1-n15Nn16O1-o18Oo32S1-p34Sp, wherein i≤5, j≤8, n≤1, o≤1, p≤1;

12C9-i13Ci1H8-j2Hj14N1-n15Nn16O1-o18Oo, wherein i≤9, j≤18, n≤1, o≤1;

12C5-i13Ci1H7-j2Hj14N1-n15Nn16O1-o18Oo, wherein i≤5, j≤7, n≤1, o≤1;

12C3-i13Ci1H3-j2Hj14N1-n15Nn16O1-o18Oo, wherein i≤3, j≤13, n≤1, o≤1;

12C4-i13Ci1H5-j2Hj14N1-n15Nn16O1-o18Oo, wherein i≤4, j≤5, n≤1, o≤1;

12C11-i13Ci1H8-j2Hj14N2-n15Nn16O1-o18Oo, wherein i≤11, j≤8, n≤2, o≤1;

12C9-i13Ci1H7-j2Hj14N1-n15Nn16O1-o18Oo, wherein i≤9, j≤7, n≤1, o≤1; and

12C5-i13Ci1H8-j2Hj14N1-n15Nn16O1-o18Oo, wherein i≤5, j≤8, n≤1, o≤1;

wherein each of i, j, n, o and p are independently an integer or 0.

In an embodiment, for example, the isotopically labeled amino acids have the formula:


wherein,gN andhN are both15N; or one ofgN andhN is15N, and one ofaC,bC,cC,dC,eC andfC is13C; or one ofgN andhN is15N, and one ofiH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH is2H; oriO is18O; or two ofaC,bC,cC,dC,eC andfC are13C; Or one ofaC,bC,cC,dC,eC andfC is13C, and one ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH is2H; or two ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH are2H.

In an embodiment, for example, the isotopically labeled amino acids have the formula:


wherein,gN andhN are both15N, andiO is18O; orgN andhN are both15N, and two ofaC,bC,cC,dC,eC andfC are13C; orgN andhN are both15N, one ofaC,bC,cC,dC,eC andfC is13C, and one ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH is2H; or one ofgN andhN is15N, one ofaC,bC,cC,dC,eC andfC is13C, andiO is18O; orgN andhN are both15N, and two ofiH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH are2H; or one ofgN andhN is15N, and three ofaC,bC,cC,dC,eC andfC are13C; or one ofgN andhN is15N, one ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH is2H, andiO is18O; or one ofgN andhN is15N, two ofaC,bC,cC,dC,eC andfC are13C, and one ofjH,kH,lH,mH,nH,oH,pH,qH,sH,tH,uH andvH is2H; or two ofaC,bC,cC,dC,eC andfC are13C, andiO is18O; or one ofgN andhN is15N, one ofaC,bC,cC,dC,eC andfC is13C, and two ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH are2H; or four ofaC,bC,cC,dC,eC andfC are13C; or one ofaC,bC,cC,dC,eC andfC is13C, one ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH is2H, andiO is18O; or one ofgN andhN is15N, and three ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH are2H; or three ofaC,bC,cC,dC,eC andfC are13C, and one ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH is2H; or two ofjH,kH, H,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH are2H, andiO is18O; or two ofaC,bC,cC,dC,eC andfC are13C, and two ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH are2H; or one ofaC,bC,cC,dC,eC andfC is13C, and three ofjH,kH,lH,mH,nH,oH,pH,qH,sH,tH,uH andvH are2H; or four ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH are2H.

In an embodiment, for example, the isotopically labeled amino acids have the formula:


wherein, two ofgN,hN,wN andxN are15N; or one ofgN,hN,wN andxN is15N, and one ofaC,bC,cC,dC,eC andfC is13C; or one ofgN,hN,wN andxN is15N, and one ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH is2H; oriO is18O; or two ofaC,bC,cC,dC,eC andfC are13C; or one ofaC,bC,cC,dC,eC andfC is13C, and one ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH is2H; or two ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH are2H.

In an embodiment, for example, the isotopically labeled amino acids have the formula


wherein, four ofgN,hN,wN andxN are15N; or three ofgN,hN,wN andxN are15N, and one ofaC,bC,cC,dC,eC andfC is13C; or three ofgN,hN,wN andxN are15N, and one ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH is2H; or two ofgN,hN,wN andxN are15N, andiO is18O; or two ofgN,hN,wN andxN are15N, and two ofaC,bC,cC,dC,eC andfC are13C; or two ofgN,hN,wN andxN are15N, one ofaC,bC,cC,dC,eC andfC is13C, and one ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH is2H; or one ofgN,hN,wN andxN is15N, one ofaC,bC,cC,dC,eC andfC is13C, andiO is18O; or two ofgN,hN,wN andxN are15N, and two ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH are2H; or one ofgN,hN,wN andxN is15N, and three ofaC,bC,cC,dC,eC andfC are13C; Or one ofhN,wN andxN is15N, one ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH is2H, andiO is18O; or one ofgN,hN,wN andxN is15N, two ofaC,bC,cC,dC,eC andfC are13C, and one ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH is2H; or two ofaC,bC,cC,dC,eC andfC are13C, andiO is18O; or one ofgN,hN,wN andxN is15N, one ofaC,bC,cC,dC,eC andfC is13C, and two ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH are2H; or four ofaC,bC,cC,dC,eC andfC are13C; or one ofaC,bC,cC,dC,eC andfC is13C, one ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH is2H, andiO is18O; or one ofgN,hN,wN andxN is15N, and three ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH are2H; or three ofaC,bC,cC,dC,eC andfC are13C, and one ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH is2H; or two ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH are2H, andiO is18O; or two ofaC,bC,cC,dC,eC andfC are13C, and two ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH are2H; or one ofaC,bC,cC,dC,eC andfC is13C, and three ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH are2H; or four ofjH,kH,lH,mH,nH,oH,pH,qH,rH,sH,tH,uH andvH are2H.

Methods of the invention include quantification approaches using isotopically encoded tagging agents, such as isotopically labeled tagging reagents, and isotopically encoded labels, such as isotopically labeled functional groups of analytes including isotopically labeled peptide groups. In an embodiment, for example, the isotopic tagging reagents comprise an amine reactive group or a carboxylic acid reactive group, such as one or more functional groups that react with an amine group or carboxylic acid group of a protein or peptide. In an embodiment, for example, the isotopic tagging reagents are isotopologues of a peptide isotopic tag or modified peptide isotopic tag. In an embodiment, for example, the isotopic tagging reagents are isotopologues of a peptide label reagent. In an embodiment, for example, the isotopologues of the peptide isotopic tag or modified peptide isotopic tag of each sample have an isotopic composition for its coded element formula selected from the group consisting of:

12C9-i13Ci1H7-j12Hj35Cl1-m37Clm14N1-n15Nn16O1-o18Oo, wherein i≤9, j≤7, m≤1, n≤1, o≤1;

12C5-i13Ci1H1-j2Hj14N5-n15Nn16O1-o18Oo, wherein i≤5, j≤1, n≤5, o≤1;

12C5-i13Ci1H6-j2Hj14N2-n15Nn, wherein i≤5, j≤6, n≤2;

12C3-i13Ci1H2-j2Hj14N5-n15Nn, wherein i≤3, j≤2, n≤5;

12C4-i13Ci1H7-i2Hj14N3-n15Nn, wherein i≤4, j≤7, n≤3;

12C4-i13Ci1H6-j2Hj14N4-n15Nn, wherein i≤4, j≤6, n≤4;

12C9-i13Ci1H7-j2Hj79Br1-lBr1-l14N1-n15Nn16O1-o18Oo, wherein i≤9, j≤7, l≤1, n≤1, o≤1;

12C4-i13Ci1H2-j2Hj14N3-n15Nn16O1-o18Oo, wherein i≤4, j≤2, n≤3, o≤1;

12C4-i13Ci1H2-j2Hj14N2-n15Nn16O2-o18Oo, wherein i≤4, j≤2, n≤2, o≤2;

12C5-i13Ci1H4-j2Hj14N2-n15Nn16O2-o18Oo, wherein i≤5, j≤4, n≤2, o≤2;

12C14-i13Ci1H14-j2Hj14N3-n15Nn16O4-o18Oo, wherein i≤14, j≤14, n≤3, o≤4;

12C9-i13Ci1H11-j2Hj14N1-n15Nn16O1-o18Oo, wherein i≤9, j≤11, n≤1, o≤1;

12C10-i13Ci1H10-j2Hj14N1-n15Nn16O2-o18Oo, wherein i≤10, j≤10, n≤1, o≤2;

12C10-i13Ci1H9-j2Hj14N3-n15Nn16O3-o18Oo, wherein i≤10, j≤9, n≤3, o≤3;

12C7-i13Ci1H7-j2Hj14N1-n15Nn16O1-o18Oo, wherein i≤7, j≤7, n≤1, o≤1;

12C11-i13Ci1H12-j2Hj14N1-n15Nn16O1-o18Oo32S1-p34Sp, wherein i≤11, j≤12, n≤1, o≤1, p≤1;

12C12-i13Ci1H17-j2Hj14N1-n15Nn16O1-o18Oo, wherein i≤12, j≤17, n≤1, o≤1, p≤1;

12C9-i13Ci1H9-j2Hj14N3-n15Nn16O1-o18Oo, wherein i≤9, j≤9, n≤2, o≤1;

12C14-i13Ci1H14-j2Hj14N3-n15Nn16O4-o18Oo, wherein i≤14, j≤14, n≤3, o≤4;

12C14-i13Ci1H14-j2Hj14N3-n15Nn16O4-o18Oo, wherein i≤14, j≤14, n≤3, o≤4;

12C12-i13Ci1H13-j2Hj14N2-n15Nn16O3-o18Oo, wherein i≤12, j≤13, n≤2, o≤3;

12C16-i13Ci1H23-j2Hj14N2-n15Nn16O4-o18Oo, wherein i≤16, j≤23, n≤2, o≤4;

12C12-i13Ci1H15-j2Hj14N2-n15Nn16O3-o18Oo, wherein i≤12, j≤15, n≤2, o≤3;

12C14-i13Ci1H19-j2Hj14N2-n15Nn16O4-o18Oo, wherein i≤14, j≤19, n≤2, o≤4;

12C11-i13Ci1H13-j2Hj14N2-n15Nn16O2-o18Oo, wherein i≤11, j≤13, n≤2, o≤2;

12C8-i13Ci1H7-j2Hj14N2-n15Nn16O2-o18Oo, wherein i≤816, j≤7, n≤2, o≤2;

12C18-i13Ci1H21-j2Hj14N4-n15Nn16O5-o18Oo, wherein i≤18, j≤21, n≤4, o≤5; and

12C18-i13Ci1H21-j2Hj14N4-n15Nn16O5-o18Oo, wherein i≤18, j≤21, n≤4, o≤5;

wherein each of i, j, l, m, n, o, and p are independently an integer or 0.

In an embodiment, for example, the isotopically labeled analytes independently comprise a peptide label. In an embodiment, for example, the peptide label of each isotopically labeled analyte has an isotopic composition for its coded element formula selected from the group consisting of:

12C14-i13Ci1H12-j2Hj14N8-n15Nn16O2-o18Oo, wherein i≤14, j≤12, n≤8, o≤1;

12C27-i13Ci1H27-j2Hj14N8-n15Nn16O4-o18Oo, wherein i≤27, j≤27, n≤8, o≤4;

12C17-i13Ci1H10-j2Hj14N6-n15Nn16O1-o18Oo, wherein i≤17, j≤10, n≤6, o≤1;

12C9-i13Ci1H10-j2Hj14N6-n15Nn16O1-o18Oo, wherein i≤9, j≤10, n≤6, o≤1;

12C30-i13Ci1H31-j2Hj14N12-n15Nn16O4-o18Oo, wherein i≤30, j≤31, n≤12, o≤4;

12C31-i13Ci1H3-j2Hj14N8-n15Nn16O6-o18Oo, wherein i≤31, j≤35, n≤8, o≤6;

12C15-i13Ci1H12-j2Hj14N8-n15Nn16O1-o18Oo, wherein i≤15, j≤12, n≤8, o≤1;

12C12-i13Ci1H8-j2Hj14N9-n15Nn16O1-o18Oo, wherein i≤12, j≤8, n≤9, o≤1;

12C11-i13Ci1H6-j2Hj14N8-n15Nn16O1-o18Oo, wherein i≤11, j≤6, n≤8, o≤1;

12C31-i13Ci1H35-j2Hj14N8-n15Nn16O4-o18Oo, wherein i≤31, j≤35, n≤8, o≤4;

12C12-i13Ci1H20-j2Hj14N2-n15Nn16O2-o18Oo, wherein i≤12, j≤20, n≤2, o≤2;

12C7-i13Ci1H13-j2Hj14N2-n15Nn16O1-o18Oo, wherein i≤7, j≤13, n≤2, o≤1; and

12C18-i13Ci1H25-j2Hj14N3-n15Nn16O3-o18Oo, wherein i≤18, j≤25, n≤3, o≤3;

wherein each of i, j, n, and o are independently an integer or 0.

In an embodiment, for example, the isotopic tagging reagents of the methods are isotopologues of a small molecule isotopic tag. In an embodiment, for example, the isotopologues of the small molecule isotopic tag of each sample have an isotopic composition for its coded element formula selected from the group consisting of:

12C9-i13Ci1H14-j2Hj14N1-n15Nn, wherein i≤9, j≤14, n≤1;

12C3-i13Ci1H9-j2Hj28Si1-q30Siq, wherein i≤3, j≤9, q≤1;

12C11-i13Ci1H7-j2Hj14N1-n15Nn32S1-p34Sp, wherein i≤11, j≤7, n≤1, p≤1;

C12-i13Ci1H16-j2Hj14N6-n15Nn16O2-o18Oo32S1-p34Sp, wherein i≤12, j≤16, n≤6, o≤2, p≤1;

12C6-i13Ci1H15-j2Hj28Si1-q30Siq, wherein i≤6, j≤15, q≤1;

12C2-i13Ci1H3-j2Hj16O2-o18Oo, wherein i≤2, j≤3, o≤2;

12C3-i13Ci16O1-o18Oo, wherein i≤3, o≤1;

12C4-i13Ci1H5-j2Hj16O2-o18Oo, wherein i≤4, j≤5, o≤2;

12C1-i13Ci1H2-j2Hj14N2-n15Nn, wherein i≤1, j≤2, n≤2;

12C6-i13Ci1H42Hj14N2-n15Nn16O2-o18Oo, wherein i≤6, j≤4, n≤2, o≤2;

12C2-i13Ci16O1-o18Oo, wherein i≤2, o≤1;

12C7-i13Ci1H6-j2Hj14N2-n15Nn16O3-o18Oo, wherein i≤7, j≤6, n≤2, o≤3;

12C7-i13Ci1H7-j2Hj14N3-n15Nn16O1-o18Oo, wherein i≤7, j≤7, n≤3, o≤1;

12C6-i13Ci1H3-j2Hj14N4-n15Nn16O4-o18Oo, wherein i≤6, j≤3, n≤4, o≤4;

12C6-i13Ci1H1-j2Hj16O2-o18Oo, wherein i≤6, j≤1, o≤2;

12C15-i13Ci1H11-j2Hj16O2-o18Oo, wherein i≤15, j≤11, o≤2;

12C6-i13Ci1H8-j2Hj16O2-o18Oo, wherein i≤6, j≤8, o≤2;

12C12-j13Ci1H12-j2Hj14N3-n15Nn16O2-o18Oo32S1-p34Sp, wherein i≤12, j≤12, n≤3, o≤2, p≤1;

12C18-i13Ci1H23-j2Hj14N2-n15Nn16O1-o18Oo, wherein i≤18, j≤23, n≤2, o≤1;

12C5-i13Ci1H4-j2Hj14N3-n15Nn, wherein i≤5, j≤4, n≤3;

12C6-i13Ci1H8-j2Hj16O2-o18Oo, wherein i≤6, j≤8, o≤2;

12C6-i13Ci1H7-j2Hj14N3-n15Nn, wherein i≤6, j≤7, n≤3;

12C6-i13Ci1H11-j2Hj14N2-n15Nn16O1-o18Oo, wherein i≤6, j≤11, n≤2, o≤1;

12C11-i13Ci1H11-j2Hj14N3-n15Nn16O1-o18Oo, wherein i≤11, j≤11, n≤3, o≤1;

12C6-i13Ci1H2-j2Hj14N3-n15Nn16O3-o18Oo, wherein i≤6, j≤2, n≤3, o≤3;

12C9-i13Ci1H10-j2Hj14N2-n15Nn32S1-p34Sp, wherein i≤9, j≤10, n≤2, p≤1;

12C11-i13Ci1H7-j2Hj14N1-n15Nn16O1-o18Oo, wherein i≤11, j≤7, n≤1, o≤1;

12C4-i13Ci16O1-o18Oo, wherein i≤4, o≤1;

12C7-i13Ci1H4-j2Hj14N2-n15Nn16O2-o18Oo32S1-p34Sp, wherein i≤7, j≤4, n≤2, o≤2, p≤1;

12C7-i13Ci1H4-j2Hj14N1-n15Nn16O4-o18Oo, wherein i≤7, j≤4, n≤1, o≤4;

12C7-i13Ci1H14-j2Hj14N1-n15Nn16O3-o18Oo, wherein i≤8, j≤14, n≤1, o≤3;

12C14-i13Ci1H14-j2Hj14N1-n15Nn16O4-o18Oo, wherein i≤14, j≤14, n≤1, o≤4;

12C4-i13Ci1H12-jHj14N2-n15Nn, wherein i≤9, j≤12, n≤2;

12C12-i13Ci1H12-j2Hj14N3-n15Nn16O2-o18Oo32S1-p34Sp, wherein i≤12, j≤12, n≤3, o≤2, p≤1;

12C12-i13Ci1H12-j2Hj14N1-n15Nn16O2-o18Oo32S1-p34Sp, wherein i≤12, j≤12, n≤1, o≤2, p≤1;

12C6-i13Ci2H4-j2Hj14N1-n15Nn16O2-o18Oo, wherein i≤6, j≤4, n≤4, o≤2;

12C6-i13Ci2H4-j2Hj14N4-n15Nn16O2-o18Oo, wherein i≤6, j≤4, n≤4, o≤1;

12C20-i13Ci1H15-j2Hj14N2-n15Nn16O1-o18Oo, wherein i≤20, j≤15, n≤2, o≤1;

12C6-i13Ci1H12-j14N2-n15Nn, wherein i≤6, j≤12, n≤2;

12C5-i13Ci1H13-j2Hj14N1-n15Nn16O1-o18Oo, wherein i≤5, j≤13, n≤1, o≤1;

12C6-i13Ci1H4-j2Hj14N1-n15Nn16O2-o18Oo, wherein i≤6, j≤14, n≤1, o≤2; and

12C8-i13Ci1H2-j2Hj14N1-n15Nn, wherein i≤8, j≤18, n≤1;

wherein each of i, j, n, o, p and q are independently an integer or 0.

Isotopically encoded compounds useful in the present methods, such as the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins, may comprise a wide range of stable isotope combinations. In an embodiment, for example, at least a portion of the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins comprises at least one12C isotope and at least one15N isotope; and at least a portion of the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins comprises at least one13C isotope and at least one14N isotope. In an embodiment, for example, at least a portion of the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins comprises at least one12C isotope and at least one2H isotope; and at least a portion of the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins comprises at least one13C isotope and at least one1H isotope. In an embodiment, for example, at least a portion of the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins comprises at least one14N isotope and at least one2H isotope; and at least a portion of the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins comprises at least one15N isotope and at least one1H isotope. In an embodiment, for example, at least a portion of the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins comprises at least one16O isotope; and at least a portion of the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins comprises at least one18O isotope. In an embodiment, for example, at least a portion of the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins comprises at least two13C,2H or15N isotopes and at least one16O isotope; and at least a portion of the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins comprises at least one18O isotope and at least at least two12C,1H or14N isotopes. In an embodiment, for example, at least a portion of the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins comprises at least two13C,2H or15N isotopes; and at least a portion of the isotopically labeled analytes, isotopic tagging reagents, isotopically labeled amino acids and/or isotopically labeled peptide or proteins comprises at least one34S isotope and at least at least two12C,1H or14N isotopes.

In an embodiment, for example, each of the isotopically labeled analytes are independently protein analytes or modified protein analytes having a different isotopic label. In an embodiment, for example, each of the isotopically labeled analytes are independently peptide analytes or modified peptide analytes having a different isotopic label. In an embodiment, for example, the isotopically labeled analytes have molecular masses selected from the range of 50 Da to 250 kDa, optionally selected from the range of 400 Da to 250 kDa, for example, for applications directed to protein and peptide analytes. In an embodiment, for example, the isotopically labeled analytes have molecular masses with 1 to 300 mDa of each other.

Methods of the invention provide an improvement on isobaric and SILAC-type quantification approaches, for example, via accessing much larger degrees for multiplexing. Enhance multiplexing in some embodiments results, at least in part, from compatibility of the methods for a large number of isotopically coded analytes, reagents, tagging agents, labels, standards, amino acids, etc. that are isotopologues that are distinguishable on the basis of mass to charge ratio using mass spectrometry analysis techniques. In an embodiment, the invention provides a multiplex method of analyzing the relative or absolute abundances of the analyte in the plurality of samples, for example a plurality of samples corresponding to difference in vivo or in vitro conditions. In an embodiment, for example, the method is for analyzing the relative or absolute abundances abundance of an analyte in at least 2 samples, optionally for some applications at least 4 samples, optionally for some applications at least 8 samples, optionally for some applications at least 20 samples. In an embodiment, for example, the step of providing the plurality of cell cultures comprises providing 2 to 20 cell cultures; and wherein the step of generating a sample for each of the cell cultures comprises generating 2 to 100 samples. In an embodiment, for example, the step of analyzing the isotopically labeled analytes for each sample using the mass spectrometry analysis technique providing the resolving power equal to or greater than 100,000 generates 2 to 150 of the independent and distinguishable mass spectrometry signals corresponding to the isotopically labeled analytes.

The present methods are compatible with a wide range of mass spectrometry techniques providing useful resolving powers, including techniques designed to probe the abundances of analytes in a plurality of samples, such as protein and peptide containing samples. In an embodiment, for example, a method of the invention further comprising the step of combining the samples characterized by a different isotopically labeled analyte prior to the step of analyzing the isotopically labeled analytes or isotopically labeled standards for each sample using the mass spectrometry analysis technique providing the resolving power equal to or greater than 100,000, thereby ensuring each sample undergoes similar sample preparation, purification, ionization, fragmentation and/or detection conditions. In an embodiment, for example, different isotopically labeled analytes or isotopically labeled standards for the plurality of samples are analyzed concurrently, for example, via purification steps and mass spectrometric analysis steps of a combination of a plurality of samples. In an embodiment, for example, the step of analyzing the isotopically labeled analytes for each sample comprises: generating one or more product ions for each of the isotopically labeled analytes, and measuring mass-to-charge ratios for at least a portion of the product ions using the mass spectrometry analysis technique providing the resolving power equal to or greater than 100,000. In an embodiment, for example, the step of analyzing the isotopically labeled analytes or isotopically labeled standards for each sample is carried out using a quadrupole ion trap, Fourier transform ion cyclotron resonance ion trap, a linear quadrupole ion trap, an orbitrap ion trap, a quadrupole mass analyzer or a time of flight mass analyzer.

In an embodiment, for example, the step of analyzing the isotopically labeled analytes or isotopically labeled standards comprises generating from the isotopically labeled analyte or isotopically labeled standards, for example, using electrospray ionization and MALDI techniques. In an embodiment, for example, the step of analyzing the isotopically labeled analytes or isotopically labeled standards comprises fragmenting ions generated from the isotopically labeled analytes or isotopically labeled standards, for example using one or more techniques selected from the group consisting of collision induced dissociation (CID), surface induced dissociation (SID), laser induced dissociation (LID), electron capture dissociation (ECD), electron transfer dissociation (ETD).

In an embodiment, for example, the method of the invention further comprises purifying proteins or peptides of the samples prior to the step of analyzing the isotopically labeled protein or peptide analytes for each sample, for example, via liquid phase chromatography (e.g., HPLC), gas phase chromatography, and/or capillary electrophoresis. In an embodiment, for example, the method of the invention further comprises frationating proteins or peptides of the samples prior to the step of analyzing the isotopically labeled protein or peptide analytes for each sample.

The methods of the present invention are useful for analyzing a variety of samples, including biological materials and samples derived from biological materials, such as biofluids, cell extracts, cell lysates, tissue extracts, etc. The methods of the present invention are useful for analyzing samples derived from in vivo biological materials. The methods of the present invention are useful for analyzing samples for proteomic analysis such as micro array samples and derived from in vitro assays. In embodiment, for example, the analyte is a protein, a peptide, a modified protein or a modified peptide. The methods of the present invention are useful for analyzing samples for analysis via gas chromatography-mass spectrometry methods, liquid chromatography-mass spectrometry methods and electrophoresis-mass spectrometry methods.

In another aspect, the invention provides a method for determining the abundance of an analyte in a sample comprising the steps of: (a) providing the sample having the analyte, wherein the analyte is a peptide or protein; (b) providing an isotopically labeled standard to the sample, wherein the analyte and the isotopically labeled standard are isotopologues; and wherein the difference of the molecular mass of the analyte and the isotopically labeled standard is less than or equal to 300 mDa; (c) analyzing the analyte and the isotopically labeled standard in the sample using a mass spectrometry analysis technique providing a resolving power equal to or greater than 100,000, thereby generating independent and distinguishable mass spectrometry signals for the analyte and the isotopically labeled standard of the sample; and (e) comparing the mass spectrometry signals for the analyte and the isotopically labeled standard of the sample, thereby determining the abundance of the analyte in the sample. As used herein, an “isotopically labeled standard” refers to an isotopically encoded compound provided to a sample to allow for absolute or relative quantification, such as an isotopically encoded peptide or protein that is provided to a sample in a known amount (e.g., having a known concentration). In an embodiment, for example, the isotopically labeled standard is an isotopically encode protein or peptide synthesized using one or more isotopically labeled amino acids, such as those provided throughout the present description. In an embodiment, the method of this aspect further comprises: (a) providing a plurality of samples, wherein each sample has the analyte; (b) providing the isotopically labeled standard to each of the samples; (c) analyzing the analyte and the isotopically labeled standard in each of the samples using a mass spectrometry analysis technique providing a resolving power equal to or greater than 100,000, thereby generating independent and distinguishable mass spectrometry signals for the analyte and the isotopically labeled standard of each sample; and (e) comparing the mass spectrometry signals for the analyte and the isotopically labeled standard of each sample, thereby determining the abundances of the analyte in the plurality of samples.

The invention also provides compositions of matter including any of the isotopically encoded compounds described herein, such as isotopically labeled amino acids, isotopically labeled standards, isotopically labeled analytes, isotopic tagging reagents, and/or isotopically labeled peptides or proteins described herein, provided in a purified state. In an embodiment, for example, the invention also provides compositions of matter including any of the isotopically encoded compounds described herein, such as isotopically labeled amino acids, isotopically labeled standards, isotopically labeled analytes, isotopic tagging reagents, and/or isotopically labeled peptides or proteins described herein, provided as an isotopically enriched composition.

Without wishing to be bound by any particular theory, there may be discussion herein of beliefs or understandings of underlying principles relating to the devices and methods disclosed herein. It is recognized that regardless of the ultimate correctness of any mechanistic explanation or hypothesis, an embodiment of the invention can nonetheless be operative and useful.

