What should I do next after completing the peptide sequence identification by mass spectrometry?
After obtaining the peptide sequence identification results from mass spectrometry, further analysis should be conducted in steps according to the research objectives (qualitative, quantitative, or functional studies). The general process is as follows:
I. Quality Control and Filtering of Results
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Check scoring metrics of database search results (such as Peptide/Protein FDR, Score, Unique peptide count).
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Exclude low-confidence proteins or results supported by only one peptide to ensure data reliability.
II. Protein Qualitative and Quantitative Organization
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If only identification is performed, organize the list of identified proteins, UniProt IDs, coverage, etc.
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If quantification is involved (Label-free, TMT, etc.), compile the protein abundance matrix for each sample and perform normalization and missing value processing.
III. Differential Analysis (if group comparison is involved)
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Use statistical tests (t-test, ANOVA, etc.) to screen for differential proteins.
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Typically set Fold Change and significance thresholds (e.g., FC≥1.5 or 2, p<0.05).
IV. Functional Annotation and Pathway Enrichment
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Use databases (GO, KEGG, Reactome) for functional classification.
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Enrichment analysis to assess the biological significance of differential proteins.
V. Further Data Mining
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Protein interaction networks (STRING, Cytoscape).
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Integration with transcriptome and metabolome data to explore regulatory mechanisms.
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