Label-free quantification
Encyclopedia
Label-free quantification is a method in mass spectrometry
that aims to determine the differential expression level of protein
s in two or more biological samples. Unlike other methods for protein quantification
, label-free quantification does not use a stable isotope
containing compound to chemically bind to and thus label the protein.
(ToF), fourier transform ion cyclotron resonance
(FTICR), or Orbitrap
mass analyzers. The high-resolution power facilitates the extraction of peptide signals on the MS1 level and thus uncouples the quantification from the identification process.
This is not true for another method of label-free quantification, spectral counting, which simply counts the number of spectra identified for a given peptide in different biological samples and then integrates the results for all measured peptides of the protein(s) that are quantified.
The computational framework of label free approach includes detecting peptides, matching the corresponding peptides across multiple LC-MS data, selecting discriminatory peptides.
Intact protein expression spectrometry (IPEx) is a label-free quantification approach in mass spectrometry under development by the analytical chemistry group at the United States Food and Drug Administration
Center for Food Safety and Applied Nutrition and elsewhere. Intact proteins are analyzed by an LCMS
instrument, usually a quadrupole
time-of-flight
in profile mode, and the full protein profile is determined and quantified using data reduction software. Early results are very encouraging. In one study, two groups of treatment replicates from mammalian samples (different organisms with similar treatment histories, but not technical replicates) show dozens of low CV protein biomarkers, suggesting that IPEx is a viable technology for studying protein expression.
While the first method, introduced above, has problems due to the identity of the peptide precursor ion that is being measured which, in high-throughput studies, could easily be a completely different peptide happening to display a similar m/z ratio and elutes at the same time or overlapping with other peptides.
The second method has problems due to the fact that the peptides are identified thus making it necessary to run an additional MS/MS scan which takes time and therefore reduces the resolution of the experiment.
Clearly, differential processing of biological samples makes it necessary to have a standard which can be used to adjust the results. Peptides that are not expected to change in their expression levels in different biological samples may be used for this purpose. However, not all peptides ionize well and therefore the choice of candidates should be done after an initial study which should only characterize the protein content of the biological samples that will be investigated.
In addition, newer hybrid mass spectrometers like LTQ OrbiTrap offer the possibility to acquire MS/MS peptide identifications in parallel to the high mass precision measurement of peptides on the MS1 level. This raises the computational challenge for the processing and integration of these two sources of information and has led to the development of novel promising quantification strategies.
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
that aims to determine the differential expression level of protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s in two or more biological samples. Unlike other methods for protein quantification
Quantitative proteomics
The aim of quantitative proteomics is to obtain quantitative information about all proteins in a sample. Rather than just providing lists of proteins identified in a certain sample, quantitative proteomics yields information about differences between samples. For example, this approach can be used...
, label-free quantification does not use a stable isotope
Stable isotope
Stable isotopes are chemical isotopes that may or may not be radioactive, but if radioactive, have half-lives too long to be measured.Only 90 nuclides from the first 40 elements are energetically stable to any kind of decay save proton decay, in theory...
containing compound to chemically bind to and thus label the protein.
Implementation
Label-free quantification is based on precursor signal intensity, which is, in most cases applied to data acquired on high mass precision spectrometers equipped with the new generation of time-of-flightTime-of-flight mass spectrometry
Time-of-flight mass spectrometry is a method of mass spectrometry in which an ion's mass-to-charge ratio is determined via a time measurement. Ions are accelerated by an electric field of known strength. This acceleration results in an ion having the same kinetic energy as any other ion that has...
(ToF), fourier transform ion cyclotron resonance
Fourier transform ion cyclotron resonance
Fourier transform ion cyclotron resonance mass spectrometry, also known as Fourier transform mass spectrometry, is a type of mass analyzer for determining the mass-to-charge ratio of ions based on the cyclotron frequency of the ions in a fixed magnetic field...
(FTICR), or Orbitrap
Orbitrap
An orbitrap is a type of mass spectrometer invented by Alexander Makarov. It consists of an outer barrel-like electrode and a coaxial inner spindle-like electrode that form an electrostatic field with quadro-logarithmic potential distribution....
mass analyzers. The high-resolution power facilitates the extraction of peptide signals on the MS1 level and thus uncouples the quantification from the identification process.
This is not true for another method of label-free quantification, spectral counting, which simply counts the number of spectra identified for a given peptide in different biological samples and then integrates the results for all measured peptides of the protein(s) that are quantified.
The computational framework of label free approach includes detecting peptides, matching the corresponding peptides across multiple LC-MS data, selecting discriminatory peptides.
Intact protein expression spectrometry (IPEx) is a label-free quantification approach in mass spectrometry under development by the analytical chemistry group at the United States Food and Drug Administration
Food and Drug Administration
The Food and Drug Administration is an agency of the United States Department of Health and Human Services, one of the United States federal executive departments...
Center for Food Safety and Applied Nutrition and elsewhere. Intact proteins are analyzed by an LCMS
Liquid chromatography-mass spectrometry
Liquid chromatography–mass spectrometry is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications which has very high...
instrument, usually a quadrupole
Quadrupole
A quadrupole or quadrapole is one of a sequence of configurations of—for example—electric charge or current, or gravitational mass that can exist in ideal form, but it is usually just part of a multipole expansion of a more complex structure reflecting various orders of complexity.-Mathematical...
time-of-flight
Time-of-flight
Time of flight describes a variety of methods that measure the time that it takes for an object, particle or acoustic, electromagnetic or other wave to travel a distance through a medium...
in profile mode, and the full protein profile is determined and quantified using data reduction software. Early results are very encouraging. In one study, two groups of treatment replicates from mammalian samples (different organisms with similar treatment histories, but not technical replicates) show dozens of low CV protein biomarkers, suggesting that IPEx is a viable technology for studying protein expression.
Detecting peptides
Typically, peptide signals are detected at the MS1 level and distinguished from chemical noise through their characteristic isotopic pattern. These patterns are then tracked across the retention time dimension and used to reconstruct a chromatographic elution profile of the mono-isotopic peptide mass. The total ion current of the peptide signal is then integrated and used as a quantitative measurement of the original peptide concentration. For each detected peptide, all isotopic peaks are first found and the charge state is then assigned.While the first method, introduced above, has problems due to the identity of the peptide precursor ion that is being measured which, in high-throughput studies, could easily be a completely different peptide happening to display a similar m/z ratio and elutes at the same time or overlapping with other peptides.
The second method has problems due to the fact that the peptides are identified thus making it necessary to run an additional MS/MS scan which takes time and therefore reduces the resolution of the experiment.
Matching corresponding peptides
In contrast to differential labelling, every biological specimen needs to be measured separately in a label-free experiment. The extracted peptide signals are then mapped across few or multiple LC-MS measurements using their coordinates on the mass to charge and retention time dimension. Data from high mass precision instruments greatly facilitate this process and increase the certainty of matching correct peptide signals across runs. In addition to the m/z dimension, the TR coordinate is used to map corresponding peptides between runs.Clearly, differential processing of biological samples makes it necessary to have a standard which can be used to adjust the results. Peptides that are not expected to change in their expression levels in different biological samples may be used for this purpose. However, not all peptides ionize well and therefore the choice of candidates should be done after an initial study which should only characterize the protein content of the biological samples that will be investigated.
Selecting discriminatory peptides
Finally, sophisticated normalization methods are used to remove systematic artefacts in the peptide intensity values between LC-MS measurements. Then, discriminatory peptides are identified by selecting the peptides whose normalized intensities are different (e.g., p-value < 0.05) among multiple groups of samples.In addition, newer hybrid mass spectrometers like LTQ OrbiTrap offer the possibility to acquire MS/MS peptide identifications in parallel to the high mass precision measurement of peptides on the MS1 level. This raises the computational challenge for the processing and integration of these two sources of information and has led to the development of novel promising quantification strategies.
See also
- Protein mass spectrometryProtein mass spectrometryProtein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Mass spectrometry is an important emerging method for the characterization of proteins. The two primary methods for ionization of whole proteins are electrospray ionization and matrix-assisted laser...
- Quantitative proteomicsQuantitative proteomicsThe aim of quantitative proteomics is to obtain quantitative information about all proteins in a sample. Rather than just providing lists of proteins identified in a certain sample, quantitative proteomics yields information about differences between samples. For example, this approach can be used...
- ICATIsotope-coded affinity tagIsotope-coded affinity tags are a gel-free method for quantitative proteomics that relies on chemical labeling reagents. These chemical probes consist of three general elements: a reactive group capable of labeling a defined amino acid side chain , an isotopically coded linker, and a tag for the...
- Isobaric labelingIsobaric labelingIsobaric labeling is a mass spectrometry strategy used in quantitative proteomics. Peptides or proteins are labeled with various chemical groups that are isobaric, or the same in mass, but which fragment during tandem mass spectrometry to yield reporter ions of different mass...
- Tandem mass tags (TMT)Tandem mass tagsTandem mass tags are chemical labels used for mass spectrometry -based quantification and identification of biological macromolecules such as proteins, peptides and nucleic acids. TMT belongs to a family of reagents referred to as isobaric mass tags...
- Isobaric tags for relative and absolute quantitation (iTRAQ)ITRAQIsobaric tags for relative and absolute quantitation are a non-gel-based technique used to quantify proteins from different sources in a single experiment. It uses isotope-coded covalent tags...
- Tandem mass tags (TMT)
- SILACSilacSILAC is a technique based on mass spectrometry that detects differences in protein abundance among samples using non-radioactive isotopic labeling. It is a popular method for quantitative proteomics.-Procedure:Two populations of cells are cultivated in cell culture...
- ICAT