Isobaric labeling is a mass spectrometry strategy used in quantitative proteomics. Peptides or proteins are labeled with chemical groups that have (at least nominally) identical mass (isobaric), but vary in terms of distribution of heavy isotopes in their structure. These tags, commonly referred to as tandem mass tags, are designed so that the mass tag is cleaved at a specific linker region upon high-energy CID (HCD) during tandem mass spectrometry yielding reporter ions of different masses. The most common isobaric tags are amine-reactive tags.[1] However, tags that react with cysteine residues and carbonyl groups have also been described.[2] These amine-reactive groups go through N-hydroxysuccinimide (NHS) reactions, which are based around three types of functional groups.[2] Isobaric labeling methods include tandem mass tags (TMT), isobaric tags for relative and absolute quantification (iTRAQ), mass differential tags for absolute and relative quantification, and dimethyl labeling.[1] TMTs and iTRAQ methods are most common and developed of these methods.[1] Tandem mass tags have a mass reporter region, a cleavable linker region, a mass normalization region, and a protein reactive group and have the same total mass.[3]
A schematic of isobaric labeling: proteins are extracted from different conditions or cell types, digested into peptides, and labeled with isobaric stable isotope tags. These tags consist of reporter, balance, and reactive regions. Lighter reporter regions are paired with heavier balance regions, such that the entire tag attached to the peptide adds the same mass shift. Therefore, after mixing, in MS1, the peptides appear as a single precursor. However, when fragmented during MS2, in addition to the normal fragment ions, the reporter regions dissociate to produce ion signals which provide quantitative information regarding the relative amount of the peptide in the samples.
A typical bottom-up proteomics workflow is described by (Yates, 2014).[2] Protein samples are enzymatically digested by a protease to produce peptides. Each digested experimental sample is derivative with a different isotopic variant of the tag from a set. The samples are mixed in typically equal ratios and analyzed simultaneously in one MS run. Since the tags are isobaric and have identical chemical properties, the isotopic variants of the tags appear as a single composite peak at the same m/z value in an MS1 scan with identical liquid chromatography (LC) retention times. During a liquid chromatography-mass spectrometry (LC-MS) analysis, the fragmented peptides produce sequence-specific product ions. These product ions are used to determine the peptide sequence and the reporter tags whose abundances reflect the relative ratio of the peptide in the samples that were combined. The use of MS/MS is required to detect the tags, therefore, unlabeled peptides are not quantified.
Isobaric labeling proteomic workflow with 4 unique reagents in a set, and 7 different biological samples combined into 2 labeling groups (plexes). As the number of samples is higher than the number of reagents, the labeling should be performed in two batches.
Advantagesedit
Explained previously by (Lee, Choe, Aggarwal, 2017).[4] A key benefit of isobaric labeling over other quantification techniques (e.g. label-free) is the multiplex capabilities and thus increased throughput potential. The ability to combine and analyze several samples simultaneously in one LC-MS run eliminates the need to analyze multiple data sets and eliminates run-to-run variation. Multiplexing reduces sample processing variability, improves specificity by quantifying the peptides from each condition simultaneously, and reduces turnaround time for multiple samples. Without multiplexing, information can be missed from run-to-run, affecting identification and quantification, as peptides selected for fragmentation on one LC-MS/MS run may not be present or of suitable quantity in subsequent sample runs. The current available isobaric chemical tags facilitate the simultaneous analysis of 2 to 11 experimental samples.
Applicationsedit
ITRAQ 8plex kit
Isobaric labeling has been successfully used for many biological applications including protein identification and quantification, protein expression profiling of normal vs abnormal states, quantitative analysis of proteins for which no antibodies are available and identification and quantification of post translationally modified proteins.[4]
^ abcBantscheff, Marcus; Lemeer, Simone; Savitski, Mikhail M.; Kuster, Bernhard (2012-09-01). "Quantitative mass spectrometry in proteomics: critical review update from 2007 to the present". Analytical and Bioanalytical Chemistry. 404 (4): 939–965. doi:10.1007/s00216-012-6203-4. ISSN 1618-2650. PMID 22772140. S2CID 21085313.
^ abcYates, John (2014). "Isobaric Labeling-Based Relative Quantification in Shotgun Proteomics". Journal of Proteome Research. 13 (12): 5293–5309. doi:10.1021/pr500880b. PMC4261935. PMID 25337643.
^Thompson, Andrew; Schäfer, Jürgen; Kuhn, Karsten; Kienle, Stefan; Schwarz, Josef; Schmidt, Günter; Neumann, Thomas; Hamon, Christian (2003). "Tandem Mass Tags: A Novel Quantification Strategy for Comparative Analysis of Complex Protein Mixtures by MS/MS". Analytical Chemistry. 75 (8): 1895–1904. doi:10.1021/ac0262560. ISSN 0003-2700. PMID 12713048. S2CID 30820837.
^ abLee, Kelvin H.; Choe, Leila H.; Aggarwal, Kunal (2006-06-01). "Shotgun proteomics using the iTRAQ isobaric tags". Briefings in Functional Genomics. 5 (2): 112–120. doi:10.1093/bfgp/ell018. ISSN 2041-2649. PMID 16772272.
^Dayon L, Hainard A, Licker V, Turck N, Kuhn K, Hochstrasser DF, Burkhard PR, Sanchez JC (2008). "Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags". Anal. Chem. 80 (8): 2921–31. doi:10.1021/ac702422x. PMID 18312001. S2CID 12362045.
^Choe L, D'Ascenzo M, Relkin NR, Pappin D, Ross P, Williamson B, Guertin S, Pribil P, Lee KH (2007). "8-plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer's disease". Proteomics. 7 (20): 3651–60. doi:10.1002/pmic.200700316. PMC3594777. PMID 17880003.
December 15, 2023
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Isobaric labeling is a mass spectrometry strategy used in quantitative proteomics Peptides or proteins are labeled with chemical groups that have at least nominally identical mass isobaric but vary in terms of distribution of heavy isotopes in their structure These tags commonly referred to as tandem mass tags are designed so that the mass tag is cleaved at a specific linker region upon high energy CID HCD during tandem mass spectrometry yielding reporter ions of different masses The most common isobaric tags are amine reactive tags 1 However tags that react with cysteine residues and carbonyl groups have also been described 2 These amine reactive groups go through N hydroxysuccinimide NHS reactions which are based around three types of functional groups 2 Isobaric labeling methods include tandem mass tags TMT isobaric tags for relative and absolute quantification iTRAQ mass differential tags for absolute and relative quantification and dimethyl labeling 1 TMTs and iTRAQ methods are most common and developed of these methods 1 Tandem mass tags have a mass reporter region a cleavable linker region a mass normalization region and a protein reactive group and have the same total mass 3 A schematic of isobaric labeling proteins are extracted from different conditions or cell types digested into peptides and labeled with isobaric stable isotope tags These tags consist of reporter balance and reactive regions Lighter reporter regions are paired with heavier balance regions such that the entire tag attached to the peptide adds the same mass shift Therefore after mixing in MS1 the peptides appear as a single precursor However when fragmented during MS2 in addition to the normal fragment ions the reporter regions dissociate to produce ion signals which provide quantitative information regarding the relative amount of the peptide in the samples Contents 1 Workflow 2 Advantages 3 Applications 4 Availability 5 ReferencesWorkflow editA typical bottom up proteomics workflow is described by Yates 2014 2 Protein samples are enzymatically digested by a protease to produce peptides Each digested experimental sample is derivative with a different isotopic variant of the tag from a set The samples are mixed in typically equal ratios and analyzed simultaneously in one MS run Since the tags are isobaric and have identical chemical properties the isotopic variants of the tags appear as a single composite peak at the same m z value in an MS1 scan with identical liquid chromatography LC retention times During a liquid chromatography mass spectrometry LC MS analysis the fragmented peptides produce sequence specific product ions These product ions are used to determine the peptide sequence and the reporter tags whose abundances reflect the relative ratio of the peptide in the samples that were combined The use of MS MS is required to detect the tags therefore unlabeled peptides are not quantified nbsp Isobaric labeling proteomic workflow with 4 unique reagents in a set and 7 different biological samples combined into 2 labeling groups plexes As the number of samples is higher than the number of reagents the labeling should be performed in two batches Advantages editExplained previously by Lee Choe Aggarwal 2017 4 A key benefit of isobaric labeling over other quantification techniques e g label free is the multiplex capabilities and thus increased throughput potential The ability to combine and analyze several samples simultaneously in one LC MS run eliminates the need to analyze multiple data sets and eliminates run to run variation Multiplexing reduces sample processing variability improves specificity by quantifying the peptides from each condition simultaneously and reduces turnaround time for multiple samples Without multiplexing information can be missed from run to run affecting identification and quantification as peptides selected for fragmentation on one LC MS MS run may not be present or of suitable quantity in subsequent sample runs The current available isobaric chemical tags facilitate the simultaneous analysis of 2 to 11 experimental samples Applications edit nbsp ITRAQ 8plex kitIsobaric labeling has been successfully used for many biological applications including protein identification and quantification protein expression profiling of normal vs abnormal states quantitative analysis of proteins for which no antibodies are available and identification and quantification of post translationally modified proteins 4 Availability editThere are two types of isobaric tags commercially available tandem mass tags TMT and isobaric tags for relative and absolute quantitation iTRAQ Amine reactive TMT are available in duplex 6 plex and 10 plex and now 11 plex sets 5 Amine reactive iTRAQ are available in 4 plex and 8 plex 6 forms References edit a b c Bantscheff Marcus Lemeer Simone Savitski Mikhail M Kuster Bernhard 2012 09 01 Quantitative mass spectrometry in proteomics critical review update from 2007 to the present Analytical and Bioanalytical Chemistry 404 4 939 965 doi 10 1007 s00216 012 6203 4 ISSN 1618 2650 PMID 22772140 S2CID 21085313 a b c Yates John 2014 Isobaric Labeling Based Relative Quantification in Shotgun Proteomics Journal of Proteome Research 13 12 5293 5309 doi 10 1021 pr500880b PMC 4261935 PMID 25337643 Thompson Andrew Schafer Jurgen Kuhn Karsten Kienle Stefan Schwarz Josef Schmidt Gunter Neumann Thomas Hamon Christian 2003 Tandem Mass Tags A Novel Quantification Strategy for Comparative Analysis of Complex Protein Mixtures by MS MS Analytical Chemistry 75 8 1895 1904 doi 10 1021 ac0262560 ISSN 0003 2700 PMID 12713048 S2CID 30820837 a b Lee Kelvin H Choe Leila H Aggarwal Kunal 2006 06 01 Shotgun proteomics using the iTRAQ isobaric tags Briefings in Functional Genomics 5 2 112 120 doi 10 1093 bfgp ell018 ISSN 2041 2649 PMID 16772272 Dayon L Hainard A Licker V Turck N Kuhn K Hochstrasser DF Burkhard PR Sanchez JC 2008 Relative quantification of proteins in human cerebrospinal fluids by MS MS using 6 plex isobaric tags Anal Chem 80 8 2921 31 doi 10 1021 ac702422x PMID 18312001 S2CID 12362045 Choe L D Ascenzo M Relkin NR Pappin D Ross P Williamson B Guertin S Pribil P Lee KH 2007 8 plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer s disease Proteomics 7 20 3651 60 doi 10 1002 pmic 200700316 PMC 3594777 PMID 17880003 Retrieved from https en wikipedia org w index php title Isobaric labeling amp oldid 1181779718, wikipedia, wiki, book, books, library,
