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Isobaric labeling

Isobaric labeling is a science topic covered in the lgStudy science library. This page brings together a partial reference excerpt, illustrations, worked examples, real-world applications and a short study plan, so you can understand Isobaric labeling rather than just read about it. In short: Isobaric labeling is a mass spectrometry strategy used in quantitative proteomics. Peptides or proteins are labeled with chemical groups that have nominally identical mass (isobaric), but vary in terms of distribution of heavy isotopes in their structure.

Isobaric labeling — main illustration
Isobaric labeling — illustration

Key takeaways

  • Isobaric labeling belongs to science; place it in that map before memorising details.
  • Learn the definition first, then one example that makes the definition concrete.
  • Connect Isobaric labeling to a quantity you can measure, compute or draw — that is where exam questions come from.
  • Reproduce the core statement of Isobaric labeling from memory before moving on to harder problems.

Reference excerpt

Isobaric labeling is a mass spectrometry strategy used in quantitative proteomics. Peptides or proteins are labeled with chemical groups that have 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 collision-induced dissociation (HCD) during tandem mass spectrometry yielding reporter ions of different masses. The most common isobaric tags are amine-reactive tags. However, tags that react with cysteine residues and carbonyl groups have also been described. These amine-reactive groups go through N-hydroxysuccinimide (NHS) reactions, which are based around three types of functional groups. 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. TMTs and iTRAQ methods are most common and developed of these methods. 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.

Workflow A typical bottom-up proteomics workflow is described by (Yates, 2014). Protein samples are enzymatically digested by a protease to produce peptides. Each digested experimental sample is derivative from a set with a different isotopic variant of the tag. 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 the MS2 analysis and upon fragmentation, each isotopic variant of the tag produces 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 combined samples. The use of MS/MS is required to detect the tags, therefore, unlabeled peptides are not quantified.

Advantages Explained previously by (Lee, Choe, Aggarwal, 2017). 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 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.

Availability There 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 10-plex, and now 11-plex sets. Amine-reactive iTRAQ are available in 4-plex and 8-plex forms.

References

Illustrations

Isobaric labeling: 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 that provide quantitative information regarding the relative amount of the peptide in the samples.
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 that provide quantitative information regarding the relative amount of the peptide in the samples.
Isobaric labeling: 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.
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.
Isobaric labeling: ITRAQ 8plex kit
ITRAQ 8plex kit

Worked examples

Example 1 — a first encounter with Isobaric labeling

Start with the simplest possible case. Write down what Isobaric labeling claims or describes in one sentence, then invent the smallest concrete situation in which that sentence is true. In science, the smallest case is usually a single object, a single equation or a single measurement. Check that every symbol or term in your sentence has a meaning in that case.

Example 2 — changing one variable

Take the situation from Example 1 and change exactly one quantity: double it, halve it, or set it to zero. Predict what should happen to Isobaric labeling before you calculate. Comparing your prediction with the result is the fastest way to find out whether you understand the idea or only the words.

Example 3 — an exam-style question

Typical questions about Isobaric labeling ask you to (a) state it precisely, (b) apply it to given data, and (c) explain a limitation. Practise writing all three answers in under five minutes; the third part is what separates a full-mark answer from an average one.

Applications of Isobaric labeling

In research
Isobaric labeling appears in science research whenever the underlying quantities have to be modelled precisely. Papers usually cite it as a starting assumption and then explore where it breaks down.
In technology and industry
Engineering practice reuses Isobaric labeling in design rules, simulations and safety margins. Knowing the idea lets you read a specification sheet and understand why the numbers look the way they do.
In the classroom
Isobaric labeling is common in secondary-school and first-year university syllabi. It links to neighbouring topics Mass spectrometry, so understanding it makes those chapters shorter.
In everyday life
Look for Isobaric labeling outside the textbook — in sport, cooking, traffic, electronics or the sky above you. An example you found yourself is remembered far longer than one you were given.
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How to study Isobaric labeling in 20 minutes

  1. Read the reference excerpt below once, without taking notes.
  2. Close the page and write down what Isobaric labeling means in your own words.
  3. Compare your version with the excerpt and mark what you missed.
  4. Work through the three examples above with pen and paper.
  5. Explain Isobaric labeling out loud to somebody else — or to Teacher Smith in the lgStudy chat.

Frequently asked questions

What is Isobaric labeling in simple terms?

Isobaric labeling is a mass spectrometry strategy used in quantitative proteomics. Peptides or proteins are labeled with chemical groups that have nominally identical mass (isobaric), but vary in terms of distribution of heavy isotopes in their structure.

Why does Isobaric labeling matter?

Because it connects several science ideas at once: it gives you a definition you can apply, a quantity you can calculate, and a way to check whether a result is plausible.

How should I study Isobaric labeling?

Read the excerpt, restate it from memory, then work through the examples and applications listed on this page. The five-step study plan above takes about twenty minutes.

What does this page cover?

It gives you a compact reference excerpt plus original lgStudy explanations, examples, applications and study material on Isobaric labeling.

Tags

  • Mass spectrometry

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