Workflow Overview for Targeted Quantitation
While targeted peptide quantitation has historically been the domain of triple-quadrupole mass spectrometers, recent advances in Thermo Scientific Orbitrap-based instruments have made them equally well-suited to quantitative experiments. On these instruments, MS1 level scans are used to monitor for the presence of the targeted species. When the mass of a known (or predicted) peptide is identified in an MS1 spectrum, the instrument triggers an MS/MS scan for confirmation of the target’s identity. The MS1 abundance of the targeted ion is used for absolute or relative quantitation based on its accurate mass. Alternatively, targeted quantitation can be done using dedicated MS/MS (or MSn) scans. In this case, target quantitation is based on the abundance of one, or several, MS/MS (or MSn) fragments. The dramatic increase in resolution and mass accuracy afforded by OrbitrapTM detection facilitates sensitive and selective quantitation at both the MS and MSn levels. Targeted peptide quantitation can be applied to both labeled and unlabeled targets.
Sample Preparation Workflow for Targeted Quantitation
Sample preparation can vary widely depending on the complexity of the samples, the expected concentration of targeted proteins, the required limit of quantitation, the dynamic range of the targets, the required sample throughput, and other factors. Fractionation of samples is often used to decrease sample complexity. Various targeted protein enrichment methods are also often used to purify and enrich low-abundance proteins. Proteins are then generally digested with trypsin or another proteolytic enzyme. The digested peptide mixture is desalted either off-line or on-line, to improve peptide ionization efficiency. Thermo Fisher Scientific provides various sample enrichment kits and products specially designed for sample preparation for mass spectrometry. Internal standards and QC products such as the Thermo Scientific Pierce
Peptide Retention Time Calibration (PRTC) mixture are available to provide the necessary metrics to determine assay performance. The PRTC mixture helps optimize and assess LC parameters, identify total peptide elution window and optimize MS parameters. Including it in quantitative samples makes it possible to monitor and normalize LC-MS performance between samples and over time.
Related Products
Peptide Retention Time Calibration Mixture
A comprehensive Mass Spectrometry Sample Preparation Handbook
Mass Spectrometry Workflow for Targeted Quantitation
Recently, high-resolution, accurate-mass detection has shown tremendous potential for targeted protein/peptide quantitation. The unmatched resolution afforded by Orbitrap technology allows for discrimination between the target ion species and very closely spaced interferences in complex sample matrices. This high selectivity greatly contributes to lowering the limits of detection and to increasing the accuracy and precision of quantitation.
The first step to developing a targeted quantitation method is to determine which peptide species are most representative of the respective targeted proteins. Care should also be given to select peptides that possess favorable properties for MS analysis. These peptides can be selected from discovery data, or from
a priori knowledge of the peptides expected to be present. Thermo Scientific Pinpoint software automates this process using previously acquired data or known target protein sequence information.
For targeted analysis in complex (i.e. whole proteome) samples, it is insufficient to rely on detection and quantitation of a single ion in the MS1 domain alone. This is because, despite the HR/AM features afforded by Orbitrap analysis, the presence of numerous isobaric peptide species can render essentially all MS1 signals ambiguous. Consequently, when a candidate signal is detected in the MS1 data, it’s identity must be confirmed by MS/MS analysis. Targeted peptides are identified and quantified using selected ion monitoring (SIM) and then confirmed with data-dependent or data-independent scheduled MS/MS. The most important advantage of SIM is improvement in LODs. This derives from the fact that in SIM, the entire Orbitrap ion capacity is devoted to a narrow range of ion masses around the target; consequently a signal that might be too faint to be well characterized from a full MS1 scan may be well characterized by a SIM scan. Typically, the complexity of biological samples requires confirmation of the target peptide’s identity using MS/MS, even when HR/AM precursor scan information is present. This is done using either a CID or HCD scan on hybrid ion trap-Orbitrap instruments or an HCD scan on hybrid quadrupole-Orbitrap instruments. The advantage of using a CID scan with ion trap detection in the hybrid instrument is the exceptional sensitivity provided by ion trap detection, which can provide high-quality spectra even for ultra-low-level targets.
Hybrid Orbitrap and Q Exactive instruments are routinely used in discovery-based proteomics experiments for both identification and relative quantitation. Using the same instruments for targeted quantitation experiments greatly streamlines the transition from discovery workflow to targeted workflow. The experimental advantages realized in discovery proteomics carry over to targeted quantitation. These include:
- Low detection limits as ions of a target peptide may be accumulated/enriched in the mass spectrometer prior to detection.
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High sensitivity MS/MS: Both CID and HCD can be used for identity confirmation, with CID in the ion trap being the most sensitive MS/MS mode, allowing target confirmation right at the detection limit of the SIM scan.
Data Analysis Workflow for Targeted Quantitation
Pinpoint™ software, part of the Thermo Scientific suite of MS software solutions, allows building targeted methods, processing acquired data, data verification, and method refinement. Pinpoint is also integrated with the Thermo Scientific Proteome Discoverer software, which enables direct transfer of discovery data into the targeted method to guide method creation, for example to restrict timed data acquisition methods to time periods where targeted peptides are known to elute, and to select product ions to used for peptide quantitation. In addition, discovery data can be used for processing verification through ion-ratio and retention-time confirmation across all samples. Pinpoint software performs data processing for all types of targeted quantitation experiments including relative and absolute quantitation, single-point and time-course studies, and quantitation curve generation for normal

and reverse curves. Pinpoint software can build methods and process data for all Thermo Scientific mass spectrometers used for proteomics studies.
For more detailed step-by-step information about quantitative analysis using Pinpoint software and to download a free 60-day demonstration version of the software, please visit the Thermo Scientific
Proteomics Software Portal.
Resources
Thermo Scientific Pinpoint Software: Accelerating targeted protein quantitation in biological research
Brochure
Thermo Scientific Proteome Discoverer: Mass Informatics Platform for Protein Scientists
Brochure
Quantifying Peptides in Complex Mixtures with High Sensitivity and Precision Using a Targeted Approach with a Hybrid Linear Ion Trap Orbitrap Mass Spectrometer
Kiyonami R, Prakash A, et al.
Application Note 500
Quantifying Peptides in Complex Mixtures with High Sensitivity and Precision Using a Targeted Approach with a Hybrid Linear Ion Trap-Orbitrap Mass Spectrometer
Kiyonami R, Zeller M, Zabrouskov V.
Application Note 557