Tools for PRM Proteomics Data Analysis

Parallel Reaction Monitoring (PRM) is a targeted protein quantification approach enabled by high-resolution and high-mass-accuracy mass spectrometry platforms. PRM has been extensively applied in cancer biomarker verification, drug target confirmation, and signaling pathway studies. In contrast to Selected Reaction Monitoring (SRM/MRM), PRM utilizes high-resolution instruments such as Orbitrap or Q-TOF systems to acquire full fragment ion spectra, thereby improving analytical selectivity and quantitative accuracy. This document provides an integrated overview of commonly used tools for PRM data analysis, including their analytical principles, distinct features, and suitable application scenarios. In addition, workflow considerations and implementation experience from Baitaipaike Biotechnology are summarized to facilitate adoption of this analytical strategy in life science research.

Overview of PRM Technology and Analytical Requirements

PRM experiments typically rely on target peptide lists generated through data-dependent acquisition (DDA) or curated based on prior knowledge. In high-resolution mass spectrometers, precursor ions are isolated at MS1 and all corresponding fragment ions are acquired at MS2, enabling specific and reproducible targeted quantification. Because PRM datasets contain chromatographic information for multiple fragment ions, downstream processing must extract ion traces, calibrate retention times, integrate peak areas, normalize quantitative values, and perform statistical comparisons. These requirements place stringent demands on analytical software, making tool selection a critical factor for successful PRM data analysis.

Mainstream Software Tools for PRM Data Analysis

1. Skyline: An Open, Flexible, And Comprehensive Analysis Environment

Skyline is a freely available open-source software developed by the MacCoss Laboratory at the University of Washington. It supports PRM, SRM, DIA, and MS1 full-scan quantification modes. Its modular design, intuitive graphical interface, and active user community have collectively positioned Skyline as the de facto standard for PRM analysis.

(1) Key Functionalities

• Supports raw data import from major instrument vendors including Thermo, SCIEX, Agilent, and Waters.

• Integrates with spectral libraries (e.g., NIST, PeptideAtlas, DIA-NN) to enhance peptide identification confidence.

• High degree of visualization, including extracted ion chromatograms (XICs) and manual peak refinement.

• Offers multiple normalization strategies (total ion intensity, internal standards, or matched peptides).

• Allows export of results in multiple formats for downstream statistical analysis (e.g., R or Python).

(2) Typical Analysis Workflow

• Import raw data files (e.g., Thermo .raw, .mzML).

• Construct a target peptide list (from DDA results, public databases, or manual curation).

• Extract fragment-ion XICs and perform peak identification.

• Conduct manual or automated integration and quantitative extraction.

• Normalize using QC peptides and generate quantitative output reports.

Skyline is widely adopted in academic research environments, particularly for studies requiring high data transparency and reproducibility.

2. Spectrodive: A High-Throughput Solution Tailored for Commercial Applications

SpectroDive is a commercial software developed by Biognosys (Switzerland) designed for PRM and MRM datasets. It offers higher levels of automation and statistical processing, making it suitable for industrial applications and large-scale multicenter validation studies.

(1) Software Advantages

• High degree of automation, enabling rapid adoption by users without programming expertise.

• UI-driven configuration of peptide libraries, internal standards, quantification rules, and statistical thresholds.

• Supports cross-sample comparisons and integrated multi-omics analysis.

• Generates publication-quality visual outputs and structured reports suitable for project delivery.

(2) Recommended Application Scenarios

• Preclinical biomarker verification studies.

• Standardized quantitative pipelines in CRO/CMO environments.

• Projects requiring rapid turnaround.

3. OpenSWATH + PyProphet: A DIA-Compatible High-Throughput Alternative

OpenSWATH was originally developed for DIA workflows; however, in combination with pyProphet for targeted peptide selection and statistical assessment, it can also be applied to PRM datasets. This combination is particularly useful for validating targets identified from DDA/DIA discovery workflows.

(1) Key Characteristics

• Open-source and modular, compatible with workflow platforms such as Nextflow and Galaxy.

• Implements scoring systems (e.g., m-score) for evaluating peptide-level confidence.

• Can be combined with TRIC for cross-batch retention-time alignment and normalization.

(2) Suitable Use Cases

• Post-screening verification of large discovery datasets.

• Exploratory analyses during early phases of project development.

• Implementation within automated data processing pipelines.

MtoZ Biolabs: Integrated PRM Data Analysis Solutions

MtoZ Biolabs leverages Thermo Orbitrap Fusion Lumos and Exploris mass spectrometry platforms in combination with Skyline-based analytical workflows to deliver end-to-end PRM analysis services from target selection to data interpretation. Key technical components of the workflow include:

1. High-Accuracy Instrument Platforms: Support MSn acquisition, polarity switching, and extended dynamic range.

2. Standardized Peptide Libraries: High-coverage and high-specificity target sets derived from multi-project experience.

3. Quality Control Framework: Incorporating stable-isotope internal standards, QC peptide monitoring, and batch-effect correction.

4. Customizable Reporting: Providing graphical summaries, statistical outputs, and structured raw data exports based on project requirements.

PRM represents a highly promising targeted quantification technology in modern proteomics and is undergoing increasing translation from academic research to industrial applications. Selection of appropriate PRM data analysis tools - whether the flexibility of Skyline, the automation of SpectroDive, or the high-throughput compatibility of OpenSWATH - directly influences data reliability and interpretability. MtoZ Biolabs combines advanced mass spectrometry instrumentation with standardized data processing to support quantitative proteomics research across academic and industrial sectors.

MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.

Related Services

MRM/PRM Quantitative Proteomics Service