Targeted proteomics:Targeted acquisition and targeted data analysis
Targeted proteomics has emerged as a powerful tool for the precise quantification of specific proteins within complex biological samples. By focusing on a predefined set of analytes, targeted proteomics offers enhanced sensitivity, specificity, and reproducibility compared to traditional, data-dependent acquisition methods. This guide will delve into the fundamental principles and practical applications of targeted proteomics, exploring both targeted acquisition and data analysis strategies at both the peptide ion (MS1) and fragment ion (MS2) levels.
1. What is targeted proteomics?
Targeted proteomics, as the name implies, is to analyze specific proteins. Compared with traditional non-targeted proteomics, it has the following characteristics:
1) Pre-set goals: Before the analysis, the researcher has already identified the protein or peptide to be studied.
2) High sensitivity and specificity: By optimizing mass spectrometry parameters and data analysis methods, high sensitivity and specific detection of target proteins can be achieved.
3) High quantitative accuracy: Targeted proteomics can provide more accurate quantitative results than non-targeted proteomics.
Targeted proteomics is a subset of mass spectrometry proteomics. Mass spectrometry proteomics is a science that uses mass spectrometry technology to study proteomes. It can be used to identify proteins, quantify proteins, analyze protein modifications, etc. There are many methods of mass spectrometry proteomics, among which targeted proteomics is one that has developed rapidly in recent years.
Application of targeted proteomics
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Biomarker discovery: Finding biomarkers for disease diagnosis and prognosis.
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Drug development: Monitoring the effect of drug treatment and studying the mechanism of drug action.
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Proteomics verification: Verifying the differential proteins discovered by non-targeted proteomics.
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Biological pathway research: Studying changes in protein expression during specific biological processes.
The rapidly evolving field of targeted proteomics has led to a diverse array of methods, often characterized by overlapping features and varying nomenclature. This complexity can obscure the fundamental principles that distinguish targeted proteomics from other mass spectrometry-based approaches. To clarify this landscape, this article categorizes targeted proteomics approaches based on two key dimensions: data acquisition strategy and ion analysis level.By combining these two dimensions, we can delineate four distinct categories of targeted proteomics methods:
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MS1 Targeted Data Acquisition: Selected Ion Monitoring (SIM) can be employed to extract targeted information from a full scan MS1 spectrum.
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MS2 Targeted Data Acquisition: This category encompasses methods like Selected Reaction Monitoring (SRM) and Multiple Reaction Monitoring (MRM), which are highly specific but limited to pre-defined analytes.
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MS1 Targeted Data Analysis: Methods such as Data-Independent Acquisition (DIA) with targeted data analysis fall into this category.
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MS2 Targeted Data Analysis: This category includes a variety of methods that leverage targeted data extraction from DIA data.
By understanding these distinctions, researchers can select the most appropriate targeted proteomics method for their specific research question. This classification framework provides a clear and concise overview of the field, facilitating informed decision-making and advancing the application of targeted proteomics in diverse scientific disciplines.
2. Targeted acquisition at peptide ion level (MS1)
Selected Ion Monitoring (SIM) is a targeted mass spectrometry technique that enhances sensitivity and selectivity by focusing on specific peptide ions. In this mode, the mass spectrometer is configured to transmit only a narrow mass-to-charge range to the detector, isolating the peptides of interest. This targeted approach significantly improves the dynamic range compared to full-scan acquisition, as background noise and interfering ions are minimized.
To enable quantification, SIM monitors the intensity of the selected peptide ion over time, generating a chromatographic peak. Traditionally, continuous monitoring of the ion was employed. However, recent advancements have shown that periodic sampling at a few data points per second is sufficient to accurately reconstruct the chromatographic peak. This intermittent sampling strategy, known as multiplexing, allows the mass spectrometer to switch between multiple peptides within a single analysis, increasing sample throughput and efficiency.
While SIM primarily provides quantitative information, additional structural confirmation can be obtained through SIM-triggered MS/MS. In this approach, if the SIM signal exceeds a predefined threshold, the instrument triggers a fragmentation event, generating MS/MS spectra for peptide identification. This hybrid technique combines the quantitative power of SIM with the qualitative capabilities of MS/MS, offering a comprehensive analytical solution.
3. Targeted data analysis at peptide ion level (MS1)
Data-independent acquisition (DIA) at the peptide level enables the acquisition of peptide data for nearly all peptide ions present in a sample. This strategy employs a high-resolution mass analyzer, such as an Orbitrap or time-of-flight, to continuously sample the full mass range at the peptide level throughout the entire chromatographic gradient. Full-range MS1 spectra, collected at regular intervals, are aligned along the time axis to generate precursor ion chromatograms through targeted data analysis. The area under the curve (AUC) of these chromatograms is subsequently calculated for peptide quantification.
MS1 DIA data can also be visualized as MS1 maps, which encapsulate mass-to-charge ratio, retention time, and intensity information for each peptide ion. However, these MS1 data lack peptide sequence information. To enable peptide quantification, retention time and mass-to-charge coordinates must be extracted using reference isotopically labeled internal standards, prior knowledge, or ad hoc MS2 spectra acquired for the most intense peptide ions or a specific set of mass-to-charge ratios defined in an inclusion list.
It's noteworthy that the data structure of this approach is akin to, or even identical to, that of screening proteomics. Quantitative data can be extracted for every peptide in the sample without any prior peptide selection. In fact, these MS1 maps have frequently been utilized for label-free quantification in screening proteomics experiments. What distinguishes this method as targeted proteomics is a subtle yet profound concept: the consistent extraction of quantitative information solely for peptides of interest, aligned with a specific hypothesis. The same acquired data, analyzed without a hypothesis and with quantitative information extracted for all available peptides, would be classified as a screening (non-targeted) proteomics experiment.
While peptide-level targeted data analysis methods can provide quantitative information for virtually any peptide precursor ion, they are susceptible to high chemical noise due to the co-elution of high- and low-abundance peptides when screening the entire mass range in complex samples. This phenomenon limits the dynamic range of these methods and often results in signals that are interfered with by other peptide ions.
4. Targeted data acquisition at fragment ion level (MS2)
4.1 Selected reaction monitoring
Selected reaction monitoring (SRM) is a powerful technique for targeted proteomics, enabling precise quantification of specific proteins. This method leverages triple quadrupole mass spectrometers to selectively isolate and monitor peptide precursor and fragment ions, resulting in highly specific and sensitive measurements.
Key steps in SRM:
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Peptide Precursor Ion Isolation: The first quadrupole mass analyzer isolates a specific peptide precursor ion using a narrow isolation window (e.g., ±1 m/z).
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Fragment Ion Generation: The isolated peptide is fragmented in the collision cell, producing a series of fragment ions.
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Fragment Ion Detection: The third quadrupole mass analyzer is set to monitor a specific fragment ion, providing a unique signature for the peptide.
Advantages of SRM:
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High Specificity: The double selection of precursor and fragment ions minimizes interference from other peptides, enhancing specificity.
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High Sensitivity: The reduced chemical noise resulting from the selective monitoring of specific ions improves sensitivity, enabling detection of low-abundance proteins.
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Quantitative Accuracy: Monitoring multiple fragment ions per peptide improves the accuracy and precision of quantification.
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Rapid Analysis: SRM can be performed rapidly, allowing for high-throughput analysis of multiple samples.
Limitations of SRM:
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Targeted Approach: SRM requires prior knowledge of the peptides of interest, limiting its application to targeted studies.
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Method Development: Developing SRM methods can be time-consuming and requires optimization of parameters such as precursor and fragment ion selection, collision energy, and dwell time.
Multiple Reaction Monitoring (MRM):
MRM is a related technique that involves monitoring multiple transitions from different peptides within a single analysis. This allows for simultaneous quantification of multiple proteins in a complex sample.
Comparison of SRM and SIM:
SRM: Offers both peptide identification and quantification, improved dynamic range, and higher sensitivity.
SIM: Provides targeted quantification of specific ions but lacks the specificity and sensitivity of SRM.
SRM is a valuable tool for targeted proteomics, providing accurate and sensitive quantification of specific proteins. By carefully selecting and monitoring appropriate transitions, researchers can gain valuable insights into protein expression and function.
4.2 Parallel reaction monitoring
Parallel Reaction Monitoring (PRM) is a targeted mass spectrometry technique that enables simultaneous analysis of all fragment ions generated from a selected peptide precursor. This approach differs from Selected Reaction Monitoring (SRM), which sequentially monitors individual fragment ions.
Key steps in PRM:
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Peptide Precursor Ion Isolation: The first quadrupole mass analyzer isolates a specific peptide precursor ion using a narrow isolation window.
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Fragment Ion Generation: The isolated peptide is fragmented in the collision cell, producing a series of fragment ions.
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Fragment Ion Detection: A high-resolution mass analyzer, such as an Orbitrap or Time-of-Flight (TOF) analyzer, is used to simultaneously detect all fragment ions.
Advantages of PRM:
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Flexibility: PRM allows for the selection of fragment ions for quantification and identification after data acquisition, providing flexibility in data analysis.
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High Resolution and Mass Accuracy: High-resolution mass analyzers enable precise mass measurement of fragment ions, improving specificity and sensitivity.
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High Dynamic Range: PRM can accommodate a wide range of peptide concentrations, making it suitable for quantitative analysis.
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High Sensitivity: The simultaneous detection of multiple fragment ions enhances the sensitivity of PRM, allowing for the detection of low-abundance proteins.
Limitations of PRM:
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Targeted Approach: PRM requires prior knowledge of the peptides of interest, limiting its application to targeted studies.
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Data Analysis Complexity: The large amount of data generated by PRM requires sophisticated data analysis tools for accurate interpretation.
PRM is a powerful technique for targeted proteomics, offering high sensitivity, specificity, and dynamic range. By combining the advantages of targeted acquisition with the flexibility of high-resolution mass spectrometry, PRM provides valuable insights into protein expression and function.
5. Targeted data analysis at fragment ion level (MS2)
Data-independent acquisition (DIA) mass spectrometry has emerged as a powerful analytical tool for comprehensive proteomic analysis. Unlike targeted methods like selected ion monitoring (SIM), precursor ion monitoring (PRM), and selected reaction monitoring (SRM), DIA acquires data for all peptides within a sample, enabling post-acquisition selection of target analytes.
DIA methods typically employ one or more broadband isolation windows to isolate and fragment all peptide ions within a specific mass range during each mass spectrometry cycle. The resulting fragment ion maps are then subjected to targeted data analysis, where peptide precursors and their corresponding fragment ions are identified based on various parameters, including retention time, relative ion intensity, spectral library matching, and co-elution with internal reference peptides.
A key characteristic of DIA methods is the flexibility of their isolation windows. These windows can vary in width, ranging from 2-3 m/z to the full mass range, and can be acquired sequentially, non-consecutively, or in an overlapping manner. This versatility allows for tailored experimental designs to address specific research questions.
DIA is commonly implemented on quadrupole-orbitrap and quadrupole-time-of-flight hybrid mass spectrometers. These instruments offer high sensitivity and mass accuracy, enabling the identification and quantification of both peptide and fragment ions with improved dynamic range compared to traditional MS1-based methods.
While DIA generates comprehensive datasets containing information on all peptides present in a sample, it is often employed in a targeted manner. This involves defining a set of target proteins or peptides of interest, such as specific protein isoforms, splice variants, or post-translationally modified peptides. The acquired data is then interrogated to extract quantitative information for these predefined targets. However, DIA's data-rich nature also allows for non-targeted analysis, enabling the discovery of unexpected proteins and modifications.
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