There are quite a few ways to quantify peptides and proteins in a mass spectrometer but we will present four commonly used general strategies.
From a sample preparation view point this is one of the simplest methods for quantitation. Two or more samples are analyzed by liquid chromatography mass spectrometry (LC-MS or LC-MS/MS if MS2 scans are employed). Data is analyzed and the features of the two or more samples are compared. One method looks at the elution profile of the peaks detected in MS1scans and uses the area under the curve to compare peaks in two different samples. Due to differences in ionization, for the most part only the same exact chemical structures in two samples can be compared. The second general category of label-free quantitation is called spectral counting. In this approach peptides are identified by MS2 scans and subsequent database searching. The frequencies of identification are used to compare the samples. For example, if twenty scans in sample one matched albumin but only ten scans in sample two matched albumin, one would conclude that albumin is twice as abundant in sample one. Both of these methods for label-free quantitation typically involve normalization methods. These approaches are popular because they are relatively easy to implement and require no extra sample preparation steps. Two of the disadvantages is that they are medium throughput in relation to other available methods and are more subject to error introduced during sample preparation.
Metabolic labeling (e.g. SILAC)
Metabolic labeling methods incorporate chemical “tags” into the samples so that they can be mixed together early in sample preparation yet still distinguished and quantified separately by the mass spectrometer. Samples are grown in media or fed a diet that has either normal or heavy isotopes incorporated into the amino acids. Samples can be mixed immediately after harvesting. When analyzed by the mass spectrometer each peptide or protein exhibits two peaks; one for the normal sample and one for the ‘heavy’ sample. The intensity of these peaks (area under the curve) can be used for quantitation. Note, that the heavy isotopes change the chemical characteristics very slightly so most important attributes other than m/z (elution time, ionization efficiency, fragmentation behavior) are only negligibly effected. These methods provide very accurate quantitation and are slightly more high throughput than label free methods. There are certain limitations with regard to what samples can be analyzed. For example, feeding humans a diet enriched in heavy isotopes is not possible/legal/ethical. And some organisms must be genetically manipulated for some applications.
Isobaric tagging (i.e. iTRAQ and TMT)
This method also involves tagging proteins or peptides with chemical tags yet allows comparison of up to eight samples at once. These tags are introduced after peptides are harvested and typically after digestion. Tags comprise three parts; a reporter group, a balance group, and a reactive group. The reactive group is used to attach the tags to peptides. The balance and reporter groups are matched such that all tags (up to 8 samples can be labeled with 8 different tags) add the sample total mass to the peptide. After labeling samples can be mixed and analyzed by mass spectrometry. In contrast to metabolic labeling each peptide produces only one peak in the MS1 spectrum. But upon fragmentation the tags fragment to produce up to eight reporter ions, the relative abundance of which can be used for quantitation. Note, fragmentation also gives rise to typical sequence informative fragment ions that can be used for identification. Isobaric tagging is popular because it allows many samples to be compared at once (thus increasing throughput) and can be applied to virtually any sample. It suffers from a unique yet widespread source of quantitative error when more than one peptide is isolated prior to fragmentation. Several methods have been proposed to address this problem but there are no publications describing a complete fix.
Selected reaction monitoring (SRM)
All of the previous methods are commonly applied shotgun proteomic analyses in which all, or most, peptides observed in MS1 scans are selected for MS2 scans. In contrast SRM is used almost exclusively in targeted proteomic studies. In these studies peptides of interest are known before running the sample and are specifically selected for MS2 scans. The mass spectrometer repeatedly isolates and fragments a single m/z regardless of whether it is detected in an MS1 scan. Selected MS2 fragment ions are monitored. The areas under the curve for the monitored fragment ions are used for quantitation. Often synthetic heavy labeled peptides are spiked into the sample in known quantities. These targeted approaches can often achieve greater sensitivity, reproducibility, and accuracy when compared to the other quantitation methods listed above. Moreover, they can be used to determine absolute quantities of peptides and proteins. However, they are much lower throughput (though the throughput is steadily increasing) and typically more expensive (per quantified peptide) than other methods.