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Orbitrap_SciLib
Reputable Mentor II
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The objective of targeted protein quantitation experiments is to determine the protein and/or peptide expression levels of known targets in biological systems. The experiment may be designed to determine either the relative levels of the target species or the absolute levels. The scope of these experiments can range from the analysis of individual samples in a research environment to the assessment of thousands of samples in a clinical research setting.  Mass spectrometry-based targeted quantitation requires a priori knowledge of the molecular targets, as well as of the general properties of the samples in which they are contained.  At a minimum, the scientist must know the molecular weight of the targeted species. Knowledge of additional properties of the targets, such as their LC elution times, their expected range of expression levels, and their dynamic range, as well as knowledge of the characteristics of the background matrix, will all help greatly in designing a successful targeted quantitation experiment.  Demand for targeted quantitation experiments among scientists and clinicianshas undergone explosive growth in recent years. 

Fortunately, advances in mass spectrometry hardware, including high-resolution, accurate-mass (HR/AM) instruments, sample preparation methodologies, and data processing software have now made it possible to develop robust targeted quantitation workflows suitable for analysis of hundreds to thousands of targets in a single analysis.

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Overview

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.

Related resources

Quantifying Peptides in Complex Mixtures with High Sensitivity and Precision Using a Targeted Approa...

Kiyonami R, Prakash A, et al.
Application Note 500
 

Quantifying Peptides in Complex Mixtures with High Sensitivity and Precision Using a Targeted Approa...

Kiyonami R, Zeller M, Zabrouskov V.
Application Note 557
 

HR/AM Targeted Peptide Quantitation on a Q Exactive MS: A Unique Combination of High Selectivity, Se...

Zhang Y, Hao Z, et al.
Application Note 554

Targeted Kinase Inhibitor Profiling Using a Hybrid Quadrupole-Orbitrap Mass Spectrometer

Peterman S, Bomgarden R, Viner R.
Application Note 574



 

Featured Literature

Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted prote...

Peterson AC, Russell JD, et al.
Mol Cell Proteomics. 2012 Nov;11(11):1475-88.
 

Targeted Proteomic Quantification on Quadrupole�Orbitrap Mass Spectrometer

Gallien S, Duriez E, et al.
Mol Cell Proteomics. 2012 Dec;11(12):1709-23.
 

Selectivity of LC-MS/MS analysis: Implication for proteomics experiments.

Gallien S, Duriez E, et al.
J Proteomics. 2012 Nov 14. pii: S1874-3919(12)00745-2.

Sample Preparation

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

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:

  • High mass accuracy: Allows for greater selectivity, accuracy and precision of quantitation at the same time increasing confidence in the identity of the quantified target.

 

  • Ultra-high resolution: Increases selectivity and lowers detection limits for targeted analyte

 

  • High intra-scan dynamic range: Increases linearity and dynamic range of the quantitation experiment

 

  • Low detection limits as ions of a target peptide may be accumulated/enriched in the mass spectrometer prior to detection.
     
  • 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.


     

Resources


Quantifying Peptides in Complex Mixtures with High Sensitivity and Precision Using a Targeted Approa...

Kiyonami R, Prakash A, et al.
Application Note 500


Quantifying Peptides in Complex Mixtures with High Sensitivity and Precision Using a Targeted Approa...

Kiyonami R, Zeller M, Zabrouskov V.
Application Note 557

PRODUCTS

 

Orbitrap Elite

Orbitrap Velos Pro

LTQ Orbitrap XL

Q Exactive
 

Thermo Scientific provides UPLC/HPLC systems that perform at low nano, micro, and high flow rate regimes to meet a wide variety of experimental needs.  Thermo Scientific EASY-nLC and Dionex UltiMate® 3000 RSLCnano LC systems use split-free designs to achieve exceptional stability and reproducibility and they easily couple to all Thermo Scientific mass spectrometers.

Data Analysis

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 relativeand 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 resear...

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 Approa...

Kiyonami R, Prakash A, et al.
Application Note 500

 

Quantifying Peptides in Complex Mixtures with High Sensitivity and Precision Using a Targeted Approa...

Kiyonami R, Zeller M, Zabrouskov V.
Application Note 557

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 resear...

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 Approa...

Kiyonami R, Prakash A, et al.
Application Note 500

 

Quantifying Peptides in Complex Mixtures with High Sensitivity and Precision Using a Targeted Approa...

Kiyonami R, Zeller M, Zabrouskov V.
Application Note 557

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Last update:
‎10-15-2021 02:13 AM
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