MRM / PRM
MRM (multiple reaction monitoring) also called SRM (selected reaction monitoring) is a targeted data acquisition where known precursor ion signatures such as precursor m/z, chromatographic retention time and specific CID-based fragment ion m/z values are used for compound detection and quantification.
MRMs are usually performed on triple quadrupole (QqQ) instruments where the first quadrupole serves as a mass selective filter open for only the selected m/z value (often within less than one unit resolution), the second as a collision quadrupole to generate CID-based fragment ions of the selected precursor m/z and the third as a second mass filter to only select one fragment ion at a time. In the context of MRM fragment ions specific for the compound of interest are called transitions. To gain greater confidence on detection and quantification usually two or more transitions are selected per precursor ion. The aforementioned procedure has to be repeated for every transition in a QqQ instrument. Modern instruments have the required speed to cycle through many precursor ions with multiple transitions within a short time window to keep up with the sharp chromatography of contemporary uHPLC-systems.
Alternatively to the classical MRM performed on QqQ instruments hybrid QqTOF-type instruments such as the TripleTOF 5600+ can perform PRM (parallel reaction monitoring) assays. In a PRM assay the first two quadrupoles basically operate equally to those in a QqQ instrument followed by monitoring all transitions of a selected precursor in a single high resolution fragment ion spectrum recorded by the TOF analyser. PRM assays provide higher confidence detection because all possible transitions are monitored. Consequently in targeted proteomics fragment ion spectra from PRM acquisitions can also be used for protein identification through software-based spectrum interpretation using database-dependent search engines. Such a hypothesis-driven approach can be used to try to verify the presence of proteins that failed to be detected by untargeted discovery approaches. Using software-tools such as Skyline a list of suitable tryptic peptides from a number of target proteins and their most likely transitions can be predicted and fed into the analysis software of the mass spectrometer for PRM analysis. Using the TripleTOF 5600 + instrument we are able to screen for roughly 20 peptides in an unscheduled MRM approach. If retention times are known many more precursors can be selected in a scheduled PRM experiment. In such a scheduled PRM assay precursors are only selected during their specific retention time windows to keep the cycle time of the mass spectrometer as low as possible.
SWATH-MS
SWATH-MS (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) is a new strategy for high throughput, label-free protein quantification (Gillet LC et al. (2012) Mol Cell Proteomics. 11(6):O111.016717.). It generates global, quantitative protein maps using data-independent acquisition of collision-induced dissociation (CID) spectra of all precursor ions. As a data-independent acquisition SWATH-MS has a greater coverage of peptide identification/quantification compared to classical discovery approaches.
Using known fingerprints of target peptides comprising precursor mass, chromatographic retention time and MRM transitions SWATH protein maps can be interrogated for targeted quantification of proteins of interest based on high resolution MRM-like signatures. SWATH acquires all MRM transitions of all precursors and thus does not require tedious assay development and allows for a more dynamic data interpretation compared to classical MRM experiments. New proteins can be added to the list of targets during the process of data interpretation without the requirement of additional data acquisition.
How does SWATH work?
The mass spectrometer does not select and isolate a specific precursor ion for CID but fragments everything within a pre-defined mass window of about 15 to 25 m/z width to acquire a single CID fragment-ion spectrum. To cover the full mass range between m/z 400-1250 the mass spectrometer sequentially acquires one full MS spectrum and about 34 CID-MS/MS spectra with isolation windows of about 20 m/z during one cycle of roughly 3.5 seconds. Theoretically fragment ions of all precursor ions detectable throughout the selected mass range and along the chromatographic elution period are recorded. Such complex CID data however, cannot be matched to peptide sequences from databases through the commonly used search engines like Mascot, SEQUEST, ProteinPilot etc. Instead SWATH MS/MS data are searched against spectral libraries which can be generated from previous discovery data of data-dependent acquisitions.
iTRAQ
iTRAQ (isobaric Tag for Relative and Absolute Quantitation) is a multiplex protein labelling for mass spectrometry-based protein quantification. It is available as 4-plex and 8-plex labels. iTRAQ reagents label primary amines (N-terminal amino group and epsilon amino group of lysines) either of intact proteins or proteolytically digested proteins. After labelling samples are pooled and further processed for mass spectrometric analysis.
The iTRAQ labels are isobaric and cannot be distinguished by MS only. All differentially labelled peptides of the same species are pooled in one peak. Upon collision induced dissociation the tags release low mass reporter fragment ions with a tag-specific mass of either m/z 113, 114, 115 … 121 for the 8-plex label. The relative peak intensities of the different reporter ions in the CID (collision induced dissociation) MS/MS spectrum are used for relative protein quantification.
ICAT
ICAT (Isotop-Coded Affinity Tagging) is a duplex protein labelling technique to differentially compare relative protein abundances of two samples directly with the mass spectrometer. The ICAT chemistry labels the cysteine residues of proteins. Each sample is labelled either with a light or a heavy label that differ in mass by 9 Da. The mass difference of light and heavy label is due to a different carbon isotope composition of both tags and therefore does not alter the chemical properties of the labelled proteins. After labelling both protein samples are pooled, digested with trypsin and further processed for mass spectrometric analysis.
The relative abundances of differentially labelled proteins can be calculated by the relative peak intensities of tryptic peptides labelled with light versus heavy tag that differentially appear in the mass spectrum with an increment of 9 Da. The ICAT tag also contains a biotin tag for reducing sample complexity by affinity purification of labelled peptides. ICAT is therefore particularly suitable for the quantification of proteins in very complex protein mixtures.
AQUA
AQUA (absolute quantification) of proteins uses synthetic stable isotope labelled proteotypic peptides as internal standards for relative and absolute quantification. The synthetic peptides have identical amino acid sequences to endogenous proteotypic (specific for only one protein) tryptic fragments of the targeted proteins. The only difference is that the C-terminal amino acid of the synthetic peptides comprises 13C and 15N heavy stable isotopes making it 8 (heavy K) or 10 Da (heavy R) heavier than the endogenous tryptic peptides. Both peptides, the synthetic and the endogenous, have identical physic-chemical properties and thus will not be separated by sample preparation or sample fractionation. However, in the mass spectrum both peptide signals will be separated due to their mass difference and the relative peak intensities can be used for accurate quantification. Since the absolute amount of the synthetic peptide is known the intensity ratio of endogenous (light) peptide versus synthetic (heavy) peptide can be used to calculate the absolute amount of endogenous peptides which allows for the absolute quantification of the corresponding protein.
We have used this approach to count lipoprotein particles by mass spectrometry and elucidate the average stoichiometry of lipoprotein associated proteins in different particle classes (von Zychlinski A, Williams M, McCormick S, Kleffmann T (2014) Absolute quantification of apolipoproteins and associated proteins on human plasma lipoproteins. J Proteomics. 25;106:181-90. doi: 10.1016/j.jprot.2014.04.030).
Other quantification methods
We perform various other stable isotope labelling and label-free methods for relative and absolute protein quantification such as:
- SILAC – stable isotope labelling by amino acids in cell culture
- Pulsed SILAC – to measure protein stability or synthesis
- Various spectral counting approaches for label-free quantification
- TOP3 for label free quantification