SequenceAssembler: A tool for protein sequence assembly from mass spectrometry data

Accurate sequencing of complete proteoforms-that is, all molecular variants generated by post-translational modification or sequence change-is essential for advancing our understanding of biological systems, guiding biopharmaceutical development, and enabling new biotechnological applications. We present SequenceAssembler (SA), a user-friendly software post-identification tool designed to assemble full-length protein sequences by integrating both peptide-spectrum matching (PSM) and de novo sequencing data. SA is compatible with widely used proteomics tools, including Novor Cloud, PEAKS Studio, and PatternLab for Proteomics, from which it efficiently aggregates input data. Our software offers an accessible and intuitive interface, enhancing its usability. We demonstrated SA's effectiveness by analyzing bovine serum albumin and the monoclonal antibody trastuzumab, each subjected to multiple enzymatic digestions followed by high-resolution mass spectrometry. The results were consistent with those of established tools like Stitch, with performance varying depending on threshold parameters. Notably, SA distinguishes itself through its user-friendly graphical interface, one-click installation, and streamlined workflow, making it an attractive solution for a wide range of proteomics researchers. The software is freely available for academic use at http://patternlabforproteomics.org/sa. SIGNIFICANCE: SequenceAssembler fills a critical gap in proteomics by unifying peptide-spectrum matching and de novo sequencing into a single assembly platform, enabling reconstruction of full-length proteins. The intuitive graphical interface lowers barriers to adoption in core laboratories, minimizing manual steps and enhancing reproducibility. By streamlining sequence assembly workflows, SequenceAssembler empowers rapid protein sequencing to broad protein research applications.