Proteoforms are the workhorses of the cell, and subtle differences between their 
amino acid sequences or post-translational modifications (PTMs) can change their 
biological function. To most effectively identify and quantify proteoforms in 
genetically diverse samples by mass spectrometry (MS), it is advantageous to 
search the MS data against a sample-specific protein database that is tailored 
to the sample being analyzed, in that it contains the correct amino acid 
sequences and relevant PTMs for that sample. To this end, we have developed 
Spritz (https://smith-chem-wisc.github.io/Spritz/), an open-source software tool 
for generating protein databases annotated with sequence variations and PTMs. We 
provide a simple graphical user interface for Windows and scripts that can be 
run on any operating system. Spritz automatically sets up and executes 
approximately 20 tools, which enable the construction of a proteogenomic 
database from only raw RNA sequencing data. Sequence variations that are 
discovered in RNA sequencing data upon comparison to the Ensembl reference 
genome are annotated on proteins in these databases, and PTM annotations are 
transferred from UniProt. Modifications can also be discovered and added to the 
database using bottom-up mass spectrometry data and global PTM discovery in 
MetaMorpheus. We demonstrate that such sample-specific databases allow the 
identification of variant peptides, modified variant peptides, and variant 
proteoforms by searching bottom-up and top-down proteomic data from the Jurkat 
human T lymphocyte cell line and demonstrate the identification of 
phosphorylated variant sites with phosphoproteomic data from the U2OS human 
osteosarcoma cell line.