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Background: Tandem mass spectrometry (MS/MS) technology has been applied to identify proteins, as an ultimate approach to confirm the original genome annotation. To be able to identify gene fusion proteins, a special database containing peptides that cross over gene fusion breakpoints is needed. Methods: It is impractical to construct a database that includes all possible fusion peptides originated from potential breakpoints. Focusing on 6259 reported and predicted gene fusion pairs from ChimerDB 2.0 and Cancer Gene Census, we for the first time created a database CanProFu that comprehensively annotates fusion peptides formed by exon-exon linkage between these pairing genes. Results: Applying this database to mass spectrometry datasets of 40 human non-small cell lung cancer (NSCLC) samples and 39 normal lung samples with stringent searching criteria, we were able to identify 19 unique fusion peptides characterizing gene fusion events. Among them 11 gene fusion events were only found in NSCLC samples. And also, 4 alternative splicing events were characterized in cancerous or normal lung samples. Conclusions: The database and workflow in this work can be flexibly applied to other MS/MS based human cancer experiments to detect gene fusions as potential disease biomarkers or drug targets. [ABSTRACT FROM AUTHOR]