Agilent G3835AA MassHunter Mass Profiler Professional Software - Application Guide

Significance of the topic

Mass spectrometry (MS) has become a cornerstone technique for high‐throughput chemical profiling in disciplines such as metabolomics, proteomics, environmental analysis, food and beverage quality control, and natural product discovery. The vast information content of MS data requires robust chemometric tools that can systematically extract, organize, and interpret subtle differences among sample groups. Agilent MassProfiler Professional (MPP) addresses this need by providing a unified platform for feature extraction, statistical analysis, compound annotation, and pathway insights.

Study objectives and overview

The Agilent MassHunter Mass Profiler Professional Application Guide describes a step‐by‐step workflow for an unbiased differential analysis experiment. It covers eight main stages: (1) preparing the experiment design, (2) acquiring MS data, (3) feature finding, (4) data import and organization, (5) initial statistical analysis, (6) compound identification, (7) project saving, and (8) advanced operations. Each stage is orchestrated through guided wizards, ensuring consistency and reproducibility.

Methodology and instrumentation

Feature extraction is performed with MassHunter Profinder or Qualitative Analysis, producing CEF files that record retention times, masses, and abundances. Non‐Agilent data can be imported via generic CSV, XLS/XLSX, or TXT formats. After import:

• Sample grouping assigns independent variables (parameters) and replicates.
• Filtering removes low‐abundance, infrequent, or irreproducible features based on abundance thresholds, frequency of occurrence, and detection flags (present/marginal/absent).
• Alignment groups like features across files using user‐defined retention time and mass tolerances.
• Normalization and baselining adjust for instrument drift and inter‐sample variability.

Statistical analysis employs principle component analysis (PCA) for QC, t-tests or ANOVA for significance testing, and fold‐change filters to highlight biologically meaningful differences. Identified features can be exported to Agilent ID Browser for compound annotation and pathway mapping.

Main results and discussion

An initial analysis yields:

• Quality control plots showing sample clustering in PCA space, highlighting outliers or batch effects.
• Lists of statistically significant features annotated with p-values, fold changes, and frequency metrics.
• Venn diagrams and heat maps illustrating feature overlap among conditions.
• Annotated compound identifications and integrated pathway analysis for biological interpretation.

Iterative filtering and re-analysis allow refinement of results and deeper insights into molecular responses.

Benefits and practical applications

MPP’s integrated workflow accelerates metabolomic and proteomic studies, enabling laboratories to:

• Standardize data handling across diverse MS platforms (GC/MS, LC/MS, CE/MS, ICP-MS).
• Perform automated class prediction for unknown sample classification.
• Integrate compound identification and pathway analysis in a single environment.
• Share and archive complete analytical projects via TAR exports.

Future trends and opportunities

Emerging developments include:

• Machine learning–driven feature selection and predictive modeling.
• Cloud‐based, collaborative workflows for large cohorts.
• Expanded reference libraries for more comprehensive compound annotation.
• Multi‐omics integration combining MS data with genomics and transcriptomics.

Conclusion

Agilent MassProfiler Professional delivers a comprehensive solution for MS‐based differential analysis, guiding users from experimental design through feature extraction, rigorous statistical evaluation, and biological interpretation. Its modular, wizard‐driven workflow ensures reproducibility and high data quality across a wide range of applications.

Instrumentation

Supported MS platforms and software integrations:

• GC/MS, LC/MS, CE/MS, and ICP-MS instruments
• Agilent MassHunter Workstation, MassHunter Qualitative Analysis, Spectrum Mill, ChemStation
• MassHunter Profinder for batch feature extraction
• Agilent ID Browser for compound identification and pathway mapping

References

• Agilent MassHunter Mass Profiler Professional Software User Manual
• Agilent Metabolomics Workflow – Discovery Workflow Guide (p/n 5990-7067EN)
• MassHunter Profinder Quick Start Guide
• BioCyc Pathway/Genome Databases (SRI International)
• KEGG Database (Kanehisa Laboratories)