A key to measuring the absolute concentration of a peptide in a complex sample is knowing with absolute certainty, that the targeted molecule is being quantified. Using the known molecular mass of the peptide of interest can lead to an incorrect assignment, as the similitude of masses over all of the peptides within a proteome database is large. While adding retention time to this equation can increase the confidence of the assignment, chromatographic profiles can shift from run to run leading to an incorrect peptide being quantitated. Monitoring a specific peptide within a complex proteome sample is conducted through either selective reaction monitoring (SRM) or MRM [17
]. In an SRM analysis, a single product ion derived from the MS/MS fragmentation of the parent ion is measured. While MRM is fundamentally similar, many product ions are measured, increasing the certainty of identification. In both SRM and MRM, the proteome mixture is fractionated using LC directly online with MS analysis. In SRM and MRM analysis, the elution time of the analytes of interest is generally known. At a designated retention time a specific mass-to-charge (m/z
) value within the first quadrupole (Q1) region of the instrument is guided into the collision cell (Q2) and subjected to collision-induced dissociation (CID). One or more of the fragments are isolated in the Q3 region and allowed to pass onto the detector. The importance of using SRM or MRM to monitor a combination of parent and/or fragment ions of a peptide, rather than simply monitoring the parent ion of the peptide, is illustrated in . The top inset shows the tandem MS spectrum of the peptide SGGGDLTLGLEPSEEEAPR (parent ion [M + 2H]2+ m/z
957.5), with three transition ions (m
914.4, 1043.5 and 1213.6) observed in the MS/MS analysis of this peptide. Single monitoring of the m
range 957.00–958.00 (the parent ion) produces a very intense peak at ~37 min. Two other less intense peaks are observed for peptides within this m/z
range at ~31 and 42 min. Based on the intensity of the signals, any investigator would be eager to select the peak at 37 min as the peptide of interest. However, when three known product ions (m/z
914.1, 1043.5 and 1213.6) resulting from CID of the parent ion (m/z
957.5) are monitored, it shows that only the peak at 31 min gives rise to these product ions. Therefore, the peak that elutes ~31 min into the chromatogram is the peptide of interest. Besides the confidence that SRM and MRM provide, ensuring that the correct peptide signal is being measured, fragment ion monitoring also excludes a considerable amount of noise from the spectrum. Excluding the noise increases the sensitivity of the measurement over what could be obtained if only the parent ions were analyzed.
Figure 1: Importance of monitoring transition ions when conducting targeted quantitative studies. The inset shows the tandem mass spectrometry (MS2) spectrum of the HER-2 peptide SGGGDLTLGLEPSEEEAPR. The bottom four spectra show peaks throughout the chromatograms (more ...)
Probably the major challenge facing targeted, quantitative proteomics is that the MS signal obtained from a peptide behaves differentially depending on the matrix it is in. For example, it is well known that the ionization efficiency of a molecule is affected by its environment. Therefore, a solution only containing the peptide of interest will undoubtedly give a much more intense MS signal than the identical peptide within serum or plasma, for example. This phenomenon is illustrated in , which shows the effect of adding increasing amounts of cell lysate on the signals obtained from a heavy isotopic version of the surrogate peptide (SGGGDLTLGLEPSEEEAPR; [M + 2H]2+ m/z 962.5) for the protein HER2 shown above. The areas of the peaks, representing three transition ions that were monitored during an MRM experiment, all show a >50% decrease when the amount of matrix added to the pure peptide is increased from 100 to 250 ng. A final addition of 1000 ng of cell lysate to the peptide solution makes each signal essentially unquantifiable. SRM and MRM methods, will play big roles in biomarker validation, however, optimization of each assay is going to be critical and most of the data will need to be determined empirically, although computational data will help in designing the starting points for these studies.
Figure 2: Effect of matrix concentration on targeting specific peptide in complex mixture using MS. Increasing amounts of cell lysates was added to a peptide internal standard and the MS signals obtained using MRM. As the amount of matrix added increased, a concomitant (more ...)
The above example shows a fundamental need for methods/strategies enabling targeting of specific proteins or peptides within complex proteomic mixtures: the requirement to create a mixture that is enriched for the molecule of interest. While this is a simple enough concept, in practice it is not trivial. The previous example shows the detrimental effect that only a small amount of lysate can have on a peptide signal. Therefore, the higher the enrichment, the better the assay will be. One of the most intriguing methods for enriching specific peptide was demonstrated by Leigh Anderson. This method termed SISCAPA (stable isotope standards and capture by anti-peptide antibodies) uses an isotopically labeled peptide internal standard (as with AQUA) that is spiked into an enzymatically digested complex proteome sample () [18
]. In most demonstrations of SISCAPA, the proteome sample has been serum or plasma. The proteome sample, containing the peptide internal standard, is then passed over a column to which an antibody targeted to the peptide(s) of interest is immobilized. After washing the column, the bound components are eluted and analyzed using MS operating in an SRM or MRM mode. Since the m
values of the peptide of interest and its stable isotope labeled internal standard is known, they can be measured directly within the chromatogram. The antibody column is then regenerated and re-used for other samples. The value in SISCAPA is that it aids in creating a mixture that is enriched for the peptide(s) of interest and enables lower abundant species to be detected since matrix effects are minimized.
Figure 3: Schematic of stable isotope standards and capture by anti-peptide antibodies (SISCAPA) for targeted quantitative proteomic analysis. In SISCAPA, the proteome from a complex sample (such as plasma) is digested into peptides. A known quantity of stable-isotope (more ...)
One of the earliest studies showing the ability to target specific peptides in a complex biofluid sample was conducted by Hunter and Anderson [19
]. This study used immunodepleted plasma in which the six most abundant proteins were removed. The sample was then analyzed by LC–MS/MS using a multiplexed MRM for tryptic peptides representing 53 high and medium abundance proteins in human plasma. The within-run coefficients of variation (CVs) for quantitating the peptides was between 2% and 22% were for 47 out of the 53 proteins that were targeted. While immunodepletion of six high abundance proteins significantly improved CVs compared with whole plasma, the targeted analytes could still be detected in raw plasma. However, immunodepletion prior to LC–MS/MS analysis resulted in more precise measurements. Proteins present at concentrations less than 1 µg/ml could be reliably quantitated using this MRM method.
Recently, Steve Carr's laboratory published an in-depth analysis describing the use of MRM to quantitate six individual proteins that had been spiked into serum samples [20
]. In this study, human female serum was spiked with five proteins derived from non-human sources and the male-specific protein, prostate-specific antigen (PSA).
Isotopically labeled internal peptide standards were used for absolute quantitation and one of the major goals was to limit the sample preparation required to quantitatively measure each of the analytes. Obviously one of the major drawbacks of using MRM is the low-throughput compared to immunoassays. While MRM methods may never match the speed of an immunoassay, nevertheless, it is important to maximize their throughput as much as possible. This study demonstrated the ability to quantitate the six proteins with sensitivities in the 1–10 ng/ml range and concentrations spanning a linear range of two orders of magnitude. The limit of quantitation (LOQ) was in the low to mid-pg/ml range. This sensitivity and LOQ was approximately three orders of magnitude better than what can be quantitated when do direct MRM/MS analysis of plasma. While one of the objectives of this study was to keep the sample preparation minimal, it was found that using a limited SCX peptide separation strategy resulted in a 10–40-fold improvement in the LOQ.