Stable Isotope Labeling Strategies

With stable isotope labeling, one sample is derivatized with a “light” version of a chemical tag while another sample is labeled with a version of the same tag that incorporates a “heavy” isotope. The samples are then mixed together and analyzed in the same experiment. Identical compounds from the different samples co-elute as pairs of peaks and can be distinguished by the mass difference between the heavy and light isotope labels. Quantitation is performed on the pairs of peaks in the MS data and identification is performed using the MS/MS fragment data. This technique eliminates much of the bias that can be introduced when comparing peaks between different experiments since the data from all samples are collected within the same experiment.
Isotope coded affinity tags ICAT and Stable Isotope Labeling by Amino Acids in Cell Culture, or SILAC, are technologies that provide a mass difference in the molecular weights of differentially labeled peptides, which increases the complexity in the MS space and limits to comparing only 2 or 3 sample types.
Tandem Mass Tags (TMT and iTRAQ) are isobaric multiplexing tags. All versions of each tag have the same molecular mass but the positions of heavy and light isotopes are adjusted in order to affect the mass of a “reporter ion” region and “balance mass” region within the compound. Since all versions of the reagent tag are identical in molecular weight, the same peptides originating from different samples will have the same mass in MS space regardless of which reporter ion is attached. Upon fragmentation, the reporter ion can be clearly distinguished, and the identity of the peptide determined from the sequence of the larger MS/MS peptide fragments, and the quantity of that peptide from each biological sample determined from the areas of each respective reporter ion peak.

TMT, Tandem Mass Tag

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There are multiple different kits available (2-plex, 6-plex, 10-plex, 16-plex and 18-plex), including bulk, so you can adjust your order to your specific experimental needs.

TMT Quantitation Overview
TMT kits from Thermo

TMT Webinar Steven Gygi HUPO 2020

Procedure summary for MS experiments with TMT Isobaric Mass Tagging Reagents

Protein extracts isolated from cells or tissues are reduced, alkylated and digested. Samples are labeled with the TMT Reagents and then mixed before sample fractionation and clean up. Labeled samples are analyzed by high resolution Orbitrap LC-MS/MS before data analysis to identify peptides and quantify reporter ion relative abundance.

Structural design of the TMT 6-plex and 10-plex (Tandem Mass Tag) Reagents
TMT reagents consist of three functional regions: the mass reporter, the mass normalizer and the reactive group. MS/MS fragmentation sites by higher energy collision dissociation (HCD) and electron transfer dissociation (ETD) are located between the mass reporter and mass normalizer. TMT reagents are labeled with 13C and 15N heavy isotopes at varying positions. The total number of isotopes is constant for all the reagents, but the distribution between the reporter and normalizer groups are different.
There are also different reactive groups available, amine reactive groups to label primary amines as in the N-terminus and Lys side chains, sulfhydryl reactive group to label Cys side chains and carbonyl reactive groups to label carbonyl containing molecules like carbohydrates, steroids etc.

The table below shows the different masses added to the peptide for each of the TMT labels as well as the reporter ion masses.

Tandem Mass Tag (TMT) ReagentsMono Mod MassAvrg Mod MassHCD Reporter Mono m/zETD Reporter Mono m/z
Amine Reactive TMT
TMT0 - 126224.152478224.2994126.127726114.127725
TMT2 - 126225.155833225.2921126.127726114.127725
TMT2 - 127 C225.155833225.2921127.131081114.127725
TMT6/10 - 126229.162932229.2634126.127726114.127725
TMT6/10 - 127 N229.162932229.2634127.124761 115.124760
TMT10 - 127 C229.162932229.2634127.131081 114.127725
TMT10 - 128 N229.162932229.2634128.128116115.124760
TMT6/10 - 128 C229.162932229.2634128.134436 116.134433
TMT6/10 - 129 N229.162932229.2634129.131471117.131468
TMT10 - 129 C229.162932229.2634129.137790116.134433
TMT10 - 130 N229.162932229.2634130.134825117.131468
TMT6/10 - 130 C229.162932229.2634130.141145118.141141
TMT6/10 - 131229.162932229.2634131.138180 119.138176
TMT11 - 131 C229.169252229.2634131.144499 118.141141
Cystein Reactive TMT
iodoTMT0 - 126324.216141324.4185126.127725114.127725
iodoTMT6 - 126329.226595329.3825126.127725114.127725
iodoTMT6 - 127 N329.226595329.3825127.124760115.124760
iodoTMT6 - 128 C329.226595329.3825128.134433116.134433
iodoTMT6 - 129 N329.226595329.3825129.131468117.131468
iodoTMT6 - 130 C329.226595329.3825130.141141118.141141
iodoTMT6 - 131329.226595329.3825131.138176119.138176
Carbonyl Reactive TMT
aminoxyTMT0 - 126296.2212296.4084126.1277114.1277
aminoxyTMT6 - 126301.2317301.3724126.1277114.1277
aminoxyTMT6 - 127 N301.2317301.3724127.1248115.1248
aminoxyTMT6 - 128 C301.2317301.3724128.1344116.1344
aminoxyTMT6 - 129 N301.2317301.3724129.1315 117.1315
aminoxyTMT6 - 130 C301.2317301.3724130.1411 118.1411
aminoxyTMT6 - 131301.2317301.3724131.1382119.1382

TMT 6-plex kit
TMT 10-plex kit
TMT 11-plex kit
iodoTMT 6-plex kit
aminoxyTMT 6-plex kit

Structural design of the TMTpro 16-plex and 18-plex (Tandem Mass Tag) Reagents
TMT reagents consist of three functional regions: the mass reporter, the mass normalizer and the reactive group. MS/MS fragmentation site by higher energy collision dissociation (HCD) is located between the mass reporter and mass normalizer. TMT reagents are labeled with 13C and 15N heavy isotopes at varying positions. The total number of isotopes is constant for all the reagents, but the distribution between the reporter and normalizer groups are different.

The table below shows the different masses added to the peptide for each of the TMTpro labels as well as the reporter ion masses.
For TMTpro 18plex analysis, use the TMTpro 16plex monoisotopic modification mass (304.2071) for database searching.

Tandem Mass Tag (TMT) ReagentsModification Mass
Modification Mass
HCD Reporter m/z
Amine Reactive TMTpro
TMTpro-zero - 126295.189592295.3773126.127726
TMTpro-16/18plex - 126304.207146304.3127126.127726
TMTpro-16/18plex - 127 N304.207146304.3127127.124761
TMTpro-16/18plex - 127 C304.207146304.3127127.131081
TMTpro-16/18plex - 128 N304.207146304.3127128.128116
TMTpro-16/18plex - 128 C304.207146304.3127128.134436
TMTpro-16/18plex - 129 N304.207146304.3127129.131471
TMTpro-16/18plex - 129 C304.207146304.3127129.13779
TMTpro-16/18plex - 130 N304.207146304.3127130.134825
TMTpro-16/18plex - 130 C304.207146304.3127130.141145
TMTpro-16/18plex - 131 N304.207146304.3127131.13818
TMTpro-16/18plex - 131 C304.207146304.3127131.1445
TMTpro-16/18plex - 132 N304.207146304.3127132.141535
TMTpro-16/18plex - 132 C304.207146304.3127132.147855
TMTpro-16/18plex - 133 N304.207146304.3127133.14489
TMTpro-16/18plex - 133 C304.207146304.3127133.15121
TMTpro-16/18plex - 134 N304.207146304.3127134.148245
TMTpro-18plex - 134 C304.207146304.3127134.154565
TMTpro-18plex - 135 N304.207146304.3127135.151600
TMTpro - sh313.231019313.2473135.151600

TMTpro 16-plex kit
TMTpro 18-plex kit
TMTpro-134C and TMTpro-135N Label Reagents

Unimod links

  • UNIMOD TMT 2-plex link
  • UNIMOD TMT 6-plex link
  • UNIMOD cysTMT 6-plex link
  • UNIMOD iodoTMT 6-plex link
  • UNIMOD TMTpro_zero link
  • UNIMOD TMTpro 16-plex link
  • UNIMOD TMTpro 18-plex link
  • UNIMOD TMTpro super heavy link


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There are multiple different kits available, including bulk etc. shop around. The iTRAQ 4-plex kits are simply called iTRAQ without the designation as to 4-plex or 8-plex, as the original kits were all 4-plex so they didn’t need to differentiate. With the inception of 8-plex they had to add the “8-plex” terminology to the kits.

iTRAQ 4-plex 4352135 from Sigma,
iTRAQ 8-plex 4381662 from Sigma,
iTRAQ Reagents from Sigma
iTRAQ Reagents from Sciex

What is the mass added to the peptide for iTRAQ 4plex and 8plex? from the FAQ's at the Sciex site.
The table below list the masses added to the peptides per amine (N-term and Lys residues) as well as the reporter fragment m/z seen by MSMS analysis.

8-plex iTRAQ reagents113114115116117118119121
Accurate mass added to peptide304.20536304.20536304.19904304.20536304.20536304.19904304.19904304.19904
Average mass added to peptide304.3074304.3074304.3081304.3074304.3074304.3081304.3081304.3081
Monoisotopic MH+ of the reporter 113.1078114.1112115.1082116.1116117.1149118.1120119.1153121.1220
4-plex iTRAQ reagents114115116117
Accurate mass added to peptide 144.105918144.099599144.102063144.102063
Average mass added to peptide 144.1680144.1688144.1544144.1544
Monoisotopic MH+ of the reporter 114.1112115.1083116.1116117.1150

Unimod links

  • UNIMOD iTRAQ 8-plex 113, 114, 116, 117 link
  • UNIMOD iTRAQ 8-plex 115, 118, 119, 121 link
  • UNIMOD iTRAQ 4-plex 114 link
  • UNIMOD iTRAQ 4-plex 115 link
  • UNIMOD iTRAQ 4-plex 116, 117 link

SILAC Metabolic Labeling Systems

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Stable isotope labeling using amino acids in cell culture (SILAC) is a powerful method to identify and quantify relative differential changes in complex protein samples. The SILAC method uses in vivo metabolic incorporation of “heavy” 13C- or 15N-labeled amino acids into proteins followed by mass spectrometry (MS) analysis for accelerated comprehensive identification, characterization and quantitation of proteins. NeuCode amino acids enable up to four samples to be multiplexed simultaneously.

Experimental information:

There are multiple different kits from different vendors available:

SILAC systems from Thermo
search SILAC to find related products from Sigma
SILAC Kits and Reagents from Cambridge Isotope Laboratories
there are likely other I missed, so do your homework..

Software tools for SILAC quantification

  • The Trans-Proteomic Pipeline (TPP) is a complete and mature suite of free and open-source software tools for MS data representation, MS data visualization, peptide identification and validation, protein identification, quantification, and annotation, data storage and mining, and biological inference. The Automated Statistical Analysis on Protein Ratio (ASAPRatio) and XPRESS software tools are part of the TPP calculates the relative abundance of proteins, such as those obtained from stable isotope labeled precursors, by reconstructing the light and heavy elution profiles of the precursor ions and determining the elution areas of each peak

  • Skyline is also being used to integrate SILAC data.
    Check Skyline Webinar 12: Isotope Labeled Standards in Skyline for more information
    You may need to define a new Isotope Label Type: Click the "Isotope label type" dropdown list in the Peptide Settings - Modifications tab, and click the <Edit list...> element. Then specify your isotope label types in a line separated list in the form that appears. Once you have your two label types, you need to make sure that Arg10, Lys8 are checked when you have "heavy" selected in the list, and Arg6, Lys4 are checked when you have "medium" selected. All of these modifications will need to be added to the "Isotope modifications" list. The "Internal standard type" list will change to a check list (from a dropdown list), which allows you to specify multiple internal standard types. You would just make sure nothing is checked in this list for a SILAC experiment, which is the equivalent to "None" in the dropdown list. For targeted proteomics experiments for sample preparations involving both AQUA labeled peptides and 15N labeled proteins, both get checked as internal standard types.

  • MaxQuant
    MaxQuant is a quantitative proteomics software package designed for analyzing large mass-spectrometric data sets. It is specifically aimed at high-resolution MS data. Several labeling techniques as well as label-free quantification are supported. MaxQuant is freely available and can be downloaded from this site.
    MaxQuant quantitative proteomics software package
  • Perseus
    for interpreting protein quantification, interaction and post-translational modification data

  • PEAKS Q (limited free trial)
    PEAKS Q SILAC Quantification Algorithms

AQUA or Absolute QUAntitation

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Absolute QUAntitation (AQUA) in targeted proteomics analyses is performed by spiking complex samples with stable isotope-labeled synthetic peptides that act as internal standards for specific peptides.
These heavy peptides are designed to be identical to tryptic peptides generated by sample digestion, so that they co-elute with the target peptide and are concomitantly analyzed by MS/MS
But AQUA-grade peptides are costly because of their high quality and purity.

There are multiple vendors that provide AQUA peptides:

AQUA peptides from Sigma
Peptides for Targeted Quantitation from Thermo
PeptiQuant Assay Kits from Cambridge Isotope Laboratories
there are likely other I missed, so do your homework..

Data analysis
You can use Skyline to estimate the absolute molecular quantities of peptides in your experiments.
Absolute Quantification Tutorial
Webinar #13: Calibrated Quantification with Skyline

Heavy labeled protein standards

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Spiking heavy labeled ApoA-I as internal standard has been shown to be useful for accurate quantitation of this protein and/or other proteins relative to ApoA-I in a biological sample in a bottom-up proteomic workflow.

There are multiple vendors that provide heavy labeled proteins:

Heavy-Labeled MS Proteins Standards from Cambridge Isotope Laboratories
SILu Prot Protein Standards for Quantitative Mass Spectrometry from Sigma
1-Step Heavy Protein IVT Kit from Thermo
there are likely other I missed, so do your homework..

Chemical labeling

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Chemical stable isotope labeling is advantageous in that it uses inexpensive reagents and is applicable to virtually any sample.


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    TMT labeling workflow
  1. Tandem Mass Tag (TMT) Labeling Workflow Renny Lan, IDeA National Resource for Proteomics Workshop for Core Directors and Staff link (pdf)

  2. TMT labeling of small amounts of peptides
  3. Sample Preparation for Relative Quantitation of Proteins Using Tandem Mass Tags (TMT) and Mass Spectrometry (MS) Erdjument-Bromage H, Huang FK, Neubert TA. Methods Mol Biol. 2018;1741:135-149. doi: 10.1007/978-1-4939-7659-1_11. link

  4. TMTpro
  5. TMTpro Reagents: A Set of Isobaric Labeling Mass Tags Enables Simultaneous Proteome-Wide Measurements Across 16 Samples Li J, Van Vranken JG, Pontano Vaites L, Schweppe DK, Huttlin EL, Etienne C, Nandhikonda P, Viner R, Robitaille AM, Thompson AH, Kuhn K, Pike I, Bomgarden RD, Rogers JC, Gygi SP, Paulo JA. Nat Methods. 2020 Apr;17(4):399-404. doi: 10.1038/s41592-020-0781-4. Epub 2020 Mar 16. link
  6. TMTpro: Design, Synthesis, and Initial Evaluation of a Proline-Based Isobaric 16-Plex Tandem Mass Tag Reagent Set Thompson A, Wölmer N, Koncarevic S, Selzer S, Böhm G, Legner H, Schmid P, Kienle S, Penning P, Höhle C, Berfelde A, Martinez-Pinna R, Farztdinov V, Jung S, Kuhn K, Pike I. Nat Methods. 2020 Apr;17(4):399-404. doi: 10.1038/s41592-020-0781-4. Epub 2020 Mar 16. link
  7. A Triple Knockout Isobaric-Labeling Quality Control Platform With an Integrated Online Database Search Gygi JP, Ramin Rad, Navarrete-Perea J, Younesi S, Gygi SP, Paulo JA. Nat Methods. 2020 Apr;17(4):399-404. doi: 10.1038/s41592-020-0781-4. Epub 2020 Mar 16. link
  8. TMTpro: Best Practices
    Tony Herren, PhD, Field Application Scientist – Proteomics/Mass Spectrometry link (pdf)

  9. SILAC
  10. Improved SILAC quantification with data independent acquisition to investigate bortezomib-induced protein degradation Pino LK, Baeza J, Lauman R, Schilling B, Garcia BA. J Proteome Res. 2021 Apr 2;20(4):1918-1927. doi: 10.1021/acs.jproteome.0c00938. Epub 2021 Mar 25. link
  11. Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M. Mol Cell Proteomics. 2002 May;1(5):376-86. doi: 10.1074/mcp.m200025-mcp200. link
  12. Comparing SILAC- and stable isotope dimethyl-labeling approaches for quantitative proteomics. Lau HT, Suh HW, Golkowski M, Ong SE. J Proteome Res. 2014 Sep 5;13(9):4164-74. doi: 10.1021/pr500630a. Epub 2014 Aug 12. link
  13. An Overview of Advanced SILAC-Labeling Strategies for Quantitative Proteomics Terzi F, Cambridge S. Methods Enzymol. 2017;585:29-47. doi: 10.1016/bs.mie.2016.09.014. Epub 2017 Jan 12. link

  14. AQUA and Heavy labeled protein standards
  15. Multiple-Reaction Monitoring-Mass Spectrometric Assays Can Accurately Measure the Relative Protein Abundance in Complex Mixtures. Hoofnagle AN, Becker JO, Oda MN, Cavigiolio G, Mayer P, Vaisar T. Clin Chem. 2012 Apr;58(4):777-81. Epub 2012 Feb 3. link

  16. Chemical labeling
  17. Multiplex Peptide Stable Isotope Dimethyl Labeling for Quantitative Proteomics Boersema PJ, Raijmakers R, Lemeer S, Mohammed S, Heck AJ. Nat Protoc. 2009;4(4):484-94. doi: 10.1038/nprot.2009.21. link
  18. Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates. Boersema PJ, Aye TT, van Veen TA, Heck AJ, Mohammed S. Proteomics. 2008 Nov;8(22):4624-32. doi: 10.1002/pmic.200800297. link
  19. Differential Stable Isotope Labeling of Peptides for Quantitation and De Novo Sequence Derivation Goodlett DR, Keller A, Watts JD, Newitt R, Yi EC, Purvine S, Eng JK, von Haller P, Aebersold R, Kolker E. Rapid Commun Mass Spectrom. 2001;15(14):1214-21. doi: 10.1002/rcm.362. link