The necessity for highly sensitive and selective detection strategies for analytes in complex human samples was recognized early by researchers interested in the detection of small molecule analytes, such as drug metabolites and hormones. In fact, the development of selective reaction monitoring (SRM) was largely pioneered by those interested in detecting these types of compounds in highly complex and typically unfractionated samples [46–51
]. The use of SRM for the robust detection of a multitude of compounds has been reported in human plasma [49
] and other complex human samples [55
]. Indeed, the use of isotope-labeled internal standards using SRM is well established for the robust and high-throughput analysis of metabolites in complex human samples. Only more recently has the proteomics field begun using the SRM technique as a selective way to detect and quantitatively monitor unique peptides from proteins of interest [57–60
]. This shift in proteomics to targeted MS methods was largely based upon the difficulty in the detection of low-abundance proteins within the context of unfractionated complex samples such as human tissue and plasma. In fact, the accurate quantitation of peptides and proteins in human plasma is incredibly challenging due to the extreme dynamic range of ~108
]. Furthermore, monitoring low-abundance proteins in human tissue biopsies, such as the heart, which combines the complexity of a heterogeneous tissue with blood contamination, can be a daunting task.
Targeted MS (LC-MS/MS in the SRM mode) approaches require prior knowledge of the analyte to be detected. However, when coupled with the discovery methods described above, targeted MS provides the sensitivity, selectivity and throughput required for the analysis of human clinical samples. Most targeted MS techniques utilize the discriminating power of quadrupole mass analyzers to select specific peptides in a complex sample mixture for detection. Peptide SRM uses ESI followed by two phases of mass selection. The first phase (in Q1) selects for the m/z of the precursor ion (charged unique peptide). Following fragmentation of the selected peptide by collision induced dissociation (CID in q2), the second phase (in Q3) selects for the m/z of fragment ion derived from the precursor ion ().
Figure 7: Peptide quantitation using LC-MS/MS in the SRM mode. Samples are digested and isotope-labeled peptide standards corresponding to unique peptides of interest are added. Both endogenous and isotope-labeled peptides are then selected for analysis using a (more ...)
The use of two mass filters in the triple quadrupole allows for the peptide analyte to be selectively detected, even if it is present in very low abundance in an unfractionated complex mixture. This level of mass selection increases sensitivity by only allowing the transmission of a small population of ions, thus minimizing chemical background noise [62
]. Additionally, SRM measurements are easily multiplexed [63
], due to the rapid duty cycle of current instrumentation (10–100 ms), facilitating hundreds of peptides to be monitored on a chromatographic time scale in a single MS run. Because SRM assays are typically optimized for peptides separated using single phase chromatography, analyses are high-throughput and rapid with minimal sample consumption (0.5–5 μg peptides/analysis) and therefore, well suited to accommodate the large sample numbers of clinical studies.
SRM data analysis involves the comparison of the area under the curve (AUC) from extracted ion chromatograms for each transition monitored. Relative changes between multiple samples can be made without the inclusion of internal standards. However, the incorporation of stable isotope-labeled internal standards allows for high precision quantitation of each unique peptide [57
]. Because the amount of internal standard peptide is known and calibrated for the linear quantitative range, the ratio between the areas under the extracted ion chromatograms of endogenous peptide and isotope-labeled peptide allows for the calculation of the absolute amount of the endogenous peptide [57
]. Quantitation of proteins using this method is based on the detection of one or more peptides from that protein [65
]. In contrast to global MS quantitation methods, targeted MS requires prior knowledge of the peptides (proteins) selected for quantitation. This distinction, along with the throughput and sensitivity, makes it ideal for hypothesis-driven queries, and for the verification and validation of those protein candidates identified using global MS analyses.
However, obstacles may arise due to the fact that specific unique peptides are used as quantitative proxys for the protein from which they are cleaved to yield a measure of the protein abundance. While this can be advantageous in many ways because it allows for separate detection and quantitation of multiple peptides from a single protein to provide information concerning protein abundance, or even potentially varied modification states [57
], it can also be limiting if the detection of multiple peptides is difficult and/or modified peptide isoforms are below the limits of detection or quantitation within the given sample matrix. While the most comprehensive read out of protein abundance and state is achieved by the detection of multiple peptides per protein, working with an unfractionated complex sample can make this extremely difficult. This challenge becomes particularly apparent due to the shortened single dimension (RP) chromatographic separation that is used for SRM measurements, which causes all peptide components to be eluted over a short time window. Massive peptide co-elution can cause significant ion suppression and decreased signal of the peptide of interest. The phenomena of ion suppression has been extensively studied by the small molecules field, but its impact on peptide detection using SRM has been less characterized [66
]. What is clear is that significant optimization of the chromatography may be necessary for the ideal detection and quantitation of each unique peptide from candidate proteins of interest.