Quantitative proteomics
Encyclopedia
The aim of quantitative proteomics is to obtain quantitative information about all protein
s 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 to compare samples from healthy and diseased patients. The methods for protein identification are identical to those used in general (i.e. qualitative) proteomics
, but include quantification as an additional dimension.
is not a good indicator of the amount of the analyte in the sample, although differences in peak intensity of the same analyte between multiple samples accurately reflect relative differences in its abundance. One approach for relative quantitation is to separately analyzing samples by MS and compare the spectra to determine peptide abundance in one sample relative to another, as in label-free quantitation strategies
. An approach for relative quantitation that is more costly and time-consuming, though less sensitive to experimental bias than label-free quantitation, entails labeling the samples with stable isotope
labels that allow the mass spectrometer to distinguish between identical proteins in separate samples. One type of label, isotopic tags, consist of stable isotopes incorporated into protein crosslinkers that causes a known mass shift of the labeled protein or peptide in the mass spectrum. Differentially labeled samples are combined and analyzed together, and the differences in the peak intensities of the isotope pairs accurately reflect difference in the abundance of the corresponding proteins.
Absolute proteomic quantitation using isotopic peptides entails spiking known concentrations of synthetic, heavy isotopologues of target peptides into an experimental sample and then performing LC-MS/MS. As with relative quantitation using isotopic labels, peptides of equal chemistry co-elute and are analyzed by MS simultaneously. Unlike relative quantitation, though, the abundance of the target peptide in the experimental sample is compared to that of the heavy peptide and back-calculated to the initial concentration of the standard using a pre-determined standard curve to yield the absolute quantitation of the target peptide.
Relative quantitation methods include:
Absolute quantitation is performed using:
MeCAT can be used in combination with element mass spectrometry ICP-MS
allowing first-time absolute quantification of the metal bound by MeCAT reagent to a protein or biomolecule. Thus it is possible to determine the absolute amount of protein down to attomol range using external calibration by metal standard solution. It is compatible to protein separation by 2D electrophoresis and chromatography in multiplex experiments. Protein identification and relative quantification can be performed by MALDI-MS/MS and ESI-MS/MS.
product. Differential staining of gels with fluorescent dyes (difference gel electrophoresis
) can also be used to highlight differences in the spot pattern.
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 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 to compare samples from healthy and diseased patients. The methods for protein identification are identical to those used in general (i.e. qualitative) proteomics
Proteomics
Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term "proteomics" was first coined in 1997 to make an analogy with...
, but include quantification as an additional dimension.
Discovery vs. targeted proteomics
Strategies to improve the sensitivity and scope of proteomic analysis often require large sample quantities and multi-dimensional fractionation, which sacrifices throughput. Alternatively, efforts to improve the sensitivity and throughput of protein quantification limit the number of peptides that can be monitored per MS run. For this reason, proteomics research is typically divided into two categories: discovery and targeted proteomics. Discovery proteomics optimizes protein identification by spending more time and effort per sample and reducing the number of samples analyzed. In contrast, targeted proteomics strategies limit the number of features that will be monitored and then optimize the chromatography, instrument tuning and acquisition methods to achieve the highest sensitivity and throughput for hundreds or thousands of samples.Relative and absolute quantitative proteomics
Mass spectrometry is not inherently quantitative because of differences in the ionization efficiency and/or detectability of the many peptides in a given sample, which has sparked the development of methods to determine relative and absolute abundance of proteins in samples. The intensity of a peak in a mass spectrumMass spectrum
A mass spectrum is an intensity vs. m/z plot representing a chemical analysis. Hence, the mass spectrum of a sample is a pattern representing the distribution of ions by mass in a sample. It is a histogram usually acquired using an instrument called a mass spectrometer...
is not a good indicator of the amount of the analyte in the sample, although differences in peak intensity of the same analyte between multiple samples accurately reflect relative differences in its abundance. One approach for relative quantitation is to separately analyzing samples by MS and compare the spectra to determine peptide abundance in one sample relative to another, as in label-free quantitation strategies
Label-free quantification
Label-free quantification is a method in mass spectrometry that aims to determine the differential expression level of proteins in two or more biological samples...
. An approach for relative quantitation that is more costly and time-consuming, though less sensitive to experimental bias than label-free quantitation, entails labeling the samples with 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...
labels that allow the mass spectrometer to distinguish between identical proteins in separate samples. One type of label, isotopic tags, consist of stable isotopes incorporated into protein crosslinkers that causes a known mass shift of the labeled protein or peptide in the mass spectrum. Differentially labeled samples are combined and analyzed together, and the differences in the peak intensities of the isotope pairs accurately reflect difference in the abundance of the corresponding proteins.
Absolute proteomic quantitation using isotopic peptides entails spiking known concentrations of synthetic, heavy isotopologues of target peptides into an experimental sample and then performing LC-MS/MS. As with relative quantitation using isotopic labels, peptides of equal chemistry co-elute and are analyzed by MS simultaneously. Unlike relative quantitation, though, the abundance of the target peptide in the experimental sample is compared to that of the heavy peptide and back-calculated to the initial concentration of the standard using a pre-determined standard curve to yield the absolute quantitation of the target peptide.
Relative quantitation methods include:
- Isotope-coded affinity tags (ICAT)
- 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)
- Label-free quantificationLabel-free quantificationLabel-free quantification is a method in mass spectrometry that aims to determine the differential expression level of proteins in two or more biological samples...
- Metal-coded tags (MeCATs)
- N-terminal labelling
- Stable isotope labeling with amino acids in cell culture (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...
)
Absolute quantitation is performed using:
- Selected Reaction Monitoring (SRM)Selected reaction monitoringSelected reaction monitoring is a method used in tandem mass spectrometry in which an ion of a particular mass is selected in the first stage of a tandem mass spectrometer and an ion product of a fragmentation reaction of the precursor ion is selected in the second mass spectrometer stage for...
MeCAT can be used in combination with element mass spectrometry ICP-MS
ICP-MS
Inductively coupled plasma mass spectrometry is a type of mass spectrometry that is highly sensitive and capable of the determination of a range of metals and several non-metals at concentrations below one part in 1012...
allowing first-time absolute quantification of the metal bound by MeCAT reagent to a protein or biomolecule. Thus it is possible to determine the absolute amount of protein down to attomol range using external calibration by metal standard solution. It is compatible to protein separation by 2D electrophoresis and chromatography in multiplex experiments. Protein identification and relative quantification can be performed by MALDI-MS/MS and ESI-MS/MS.
When to use relative or absolute quantitation strategies
Experimental bias can influence the decision to use relative or absolute quantitation strategies. One source of bias is the mass spectrometer itself, which has a limited capacity to detect low-abundance peptides in samples with a high dynamic range. Additionally, the limited duty cycle of mass spectrometers restricts the number of collisions per unit of time, which may result in an undersampling of complex proteomic samples . Another source of bias is variation in sample preparation between experiments or individual samples in single experiments. The greater the number of steps between labeling and sample combination, the greater is the risk of introducing experimental bias. For example, during metabolic labeling, proteins are labeled in live animals or cells and the samples are then immediately combined. Because all subsequent sample preparation and analysis is performed with the combined samples, metabolic labeling has the lowest risk of experimental variation. Conversely, samples that are individually processed and analyzed in label-free quantitation strategies have a greater risk of sample variation and experimental bias.Two-dimensional gel electrophoresis
Modern day gel electrophoresis research often leverages software-based image analysis tools primarily to analyze bio-markers by quantifying individual, as well as showing the separation between one or more protein "spots" on a scanned image of a 2-DETwo-dimensional gel electrophoresis
Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins...
product. Differential staining of gels with fluorescent dyes (difference gel electrophoresis
Difference gel electrophoresis
'Difference gel electrophoresis' is a form of gel electrophoresis where up to three different protein samples can be labeled with fluorescent dyes prior to two-dimensional electrophoresis. After the gel electrophoresis, the gel is scanned with the excitation wavelength of each dye one after the...
) can also be used to highlight differences in the spot pattern.
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...
- Additional quantitative proteomics information and figures